CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 15/863,005, filed Jan. 5, 2018, which claims the benefit of U.S. Provisional Application No. 62/443,401, filed Jan. 6, 2017, both of which are incorporated herein by reference in their entireties.
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH The invention was made with Government support under contract OD017887 awarded by the National Institutes of Health. The Government has certain rights in the invention.
SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created Oct. 6, 2021, is named 079445-1273450-006220US_SL.txt and is 1.47 MB (1,547,157) bytes in size.
FIELD Provided herein are compositions and methods for identifying and using stem cell differentiation regulation factors. For example, in some embodiments, provided herein are compositions and methods for identifying stem cell differentiation regulation factors using marker gene expression libraries. Also provided herein are compositions and methods for generating differentiated and induced cells lines and uses of such cell lines.
BACKGROUND Stem cells are cells that are capable of differentiating into many cell types. Embryonic stem cells are derived from embryos and are potentially capable of differentiation into all of the differentiated cell types of a mature body. Certain types of stem cells are “pluripotent,” which refers to their capability of differentiating into many cell types. One type of pluripotent stem cell is the human embryonic stem cell (hESC), which is derived from a human embryonic source. Human embryonic stem cells are capable of indefinite proliferation in culture, and therefore, are an invaluable resource for supplying cells and tissues to repair failing or defective human tissues in vivo.
Similarly, induced pluripotent stem (iPS) cells, which may be derived from non-embryonic sources, can proliferate without limit and differentiate into each of the three embryonic germ layers. It is understood that iPS cells behave in culture essentially the same as ESCs. Human iPS cells and ES cells express one or more pluripotent cell-specific markers, such as Oct-4, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and Nanog (Yu et al. Science, Vol. 318. No. 5858, pp. 1917-1920 (2007); herein incorporated by reference in its entirety). Also, recent findings of Chan, indicate that expression of Tra 1-60, DNMT3B, and REX1 can be used to positively identify fully reprogrammed human iPS cells, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT, and NANOG are insufficient as markers of fully reprogrammed human iPS cells. (Chan et al., Nat. Biotech. 27:1033-1037 (2009); herein incorporated by reference in its entirety).
The cell fate decision making of stem cells is governed by multistep dynamic processes, in which transcriptional networks play a critical role (Chambers and Tomlinson, 2009 Development 136, 2311-2322; Filipczyk et al., 2015 Nat. Cell Biol. 17, 1235-1246; Kim et al., 2008 Cell 132, 1049-1061; MacArthur et al., 2009 Nat. Rev. Mol. Cell Biol. 10, 672-681). Expression of different transcription factors coordinate to activate or suppress sets of genes specific to different lineages, serving as major regulators that maintain cell identities or drive cell fate transitions (Iwafuchi-Doi and Zaret, 2014 Genes Dev. 28, 2679-2692; Zaret and Carroll, 2011 Genes Dev. 25, 2227-2241). The successes of somatic cell reprogramming and directed lineage differentiation using transcription factors highlight their central role in cell fate determination (Davis et al., 1987 Cell 51, 987-1000; Takahashi and Yamanaka, 2006 Cell 126, 663-676; Vierbuchen et al., 2010 Nature 463, 1035-1041; Xu et al., 2015 Cell Stem Cell 16, 119-134). Over the past few decades, although individual or combinatorial transcription factors have been identified for cell differentiation, there is a dearth of systematically unbiased studies of how specific genetic programs determine cell fate maintenance and transitions. Because of this, the available tools to control stem cell differentiation are limited and the full promise of stem cells as therapeutic, drug screening, and research tools have gone unmet.
A systematic screening approach to profile and characterize all transcription factors is needed to offer new insights into their contributions to cell fate decisions, which greatly enhances the ability to manipulate cell fate for both basic research and therapeutic purposes.
SUMMARY Provided herein are compositions and methods for identifying and using stem cell differentiation regulation factors. For example, in some embodiments, provided herein are compositions and methods for identifying stem cell differentiation regulation factors using marker gene expression libraries. Also provided herein are compositions and methods for generating differentiated and induced cells lines and uses of such cell lines.
The compositions, systems, kits, and methods of the present disclosure overcome limitations of existing technologies to identify transcription factors and nucleic that drive differentiation of pluripotent cells. The transcription factors identified using the described methods find use in research, screening, and therapeutic applications.
In some embodiments, provided herein are systems and methods for identifying factors involved in (e.g., that regulate or control) the differentiation of stem cells by employing a CRISPR activation (CRISPRa)-mediated gain-of-function screening platform. In some such embodiments, a reporter stem cell line is generated that comprises components of a CRSIPR activation system. In some embodiments, the cell line is exposed to an sgRNA library targeting all putative transcription factors or other candidate factors that may be involved in a cellular differentiation process.
In some embodiments, the CRISPR activation system comprises a dCas9 construct under the transcriptional control of a first promoter. In some embodiments, the dCas9 is fused to a peptide epitope. In some embodiments, the activation system further comprises a VP64 transactivation domain under the transcriptional control of a second promoter. In some embodiments, the VP64 transactivation domain is fused to a peptide that specifically binds to the peptide epitope. In some embodiments, the activation system further comprises a selection marker under the transcriptional control of a third promoter. In some embodiments, each of the first, second, and third promoters are different than each other.
For example, in some embodiments, provided herein is a method of identifying pluripotent cell differentiation markers, comprising: a) generating a pluripotent cell line that expresses i) nuclease dead Cas9 fused to a plurality of peptide epitopes; ii) a single chain variable chain antibody fragment specific for the peptide epitope fused to a VP64 tranactivator domain; and iii) a transactivator polypeptide; b) contacting the cell line with a plurality of single guide RNAs (sgRNAs) specific for activation of pluripotent cell differentiation factors to generate a gene activation library; c) sorting the library to identify pluripotent cells that retain pluripotency or differentiate; and d) identifying cell differentiation factors that induce or prevent differentiation of the pluripotent cells. In some embodiments, the differentiation factors are transcription factors or non-coding (e.g., lincRNAs). In some embodiments, the cells are further contacted with a plurality of non-targeting sgRNAs (e.g., to serve as a negative control). In some embodiments, the cells further overexpress endogenous POU domain, class 3, transcription factor 2 (Brn2). In some embodiments, each cell differentiation factors is targeted with a plurality (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) distinct sgRNAs. In some embodiments, the cells that retain pluripotency are identified by screening for expression of SSEA1 after culture in media lacking inhibitors of GSK3 and ERK pathways. In some embodiments, cells that differentiate are identified by expression of a differentiation marker. For example, in some embodiments, cells that differentiate into neuronal cells express Tuj1. In some embodiments, the identifying comprises sequencing of sgRNAs after selection for cells that retain pluripotency or differentiate. In some embodiments, the sequencing further comprises comparing the level of the sgRNAs to the level of non-targeting sgRNAs. In some embodiments, cell differentiation factors that retain pluripotency are one or more of the regulation factors shown in FIG. 3 or Table 3. In some embodiments, cell differentiation factors that are associated with differentiation into neuronal cells are one or more of the regulation factors shown in FIG. 6 or Tables 4 and 10. In some embodiments, the sgRNAs are dual-sgRNA-constructs comprising two sgRNAs. In some embodiments, the method further comprises contacting the cell differentiation factors with a fibroblast cell line and identifying cell differentiation factors that promote transdifferentiation of the fibroblast cell line. In some embodiments, the fibroblast cell line is contacted with combinations of two or more cell differentiation factors. In some embodiments, the cell differentiation factors that promote transdifferentiation are a combination of Ezh2 or Ngn1 and one or more additional markers (e.g., Ngn1+Brn2, Brn2+Ezh2, Mecom+Ezh2, Ngn1+Ezh2 or Ngn1+Foxo1).
In some embodiments, the pluripotent cells are induced pluripotent stem cells, adult stem cells, or embryonic stem cells. In some embodiments, the method further comprises the step of activating pairs or groups of pluripotent cell differentiation factors.
In some embodiments, the method comprises or further comprises the step of performing a CRISPR gene repression screen. For example, in some embodiments, the CRISP repression screen comprises: a) contacting a pluripotent cell that expresses dCas9 fused to a transcription repressor domain with a plurality of sgRNAs specific for repression of a plurality of cell differentiation factors; b) sorting the library to identify cells that retain pluripotency or differentiate; and c) identifying cell differentiation factors that induce or prevent differentiation of said pluripotent cells. In some embodiments, the CRISPR repression screen and the CRISPR activation screen are performed in the same or different pluripotent cells. In some embodiments, the CRISPR repression screen and the CRISPR activation screen are performed simultaneously using vectors comprising a first sgRNA specific for activation of a first cell differentiation factor and a second sgRNA specific for repression of a second cell differentiation factor.
Further embodiments provide a library of pluripotent cells generated by the methods descried herein.
Additional embodiments provide a kit or system, comprising: a) a pluripotent cell line that expresses i) nuclease dead Cas9 fused to a plurality of peptide epitopes; ii) a single chain variable chain antibody fragment specific for the peptide epitope fused to a VP64 tranactivator domain; and iii) a transactivator polypeptide; and b) a plurality of single guide RN As (sgRNAs) specific for activation of pluripotent cell differentiation factors. In some embodiments, the kit or system further comprises reagents for analysis of one or more properties (e.g., pluripotency or differentiation) of the cell lines. In some embodiments, the kit or system further comprises reagents for sequencing the cells to identify the presence of said sgRNAs. In some embodiments, the system comprises or further comprises a CRISPR repression system as described herein. In some embodiments, the system comprises one or more sgRNAs (e.g., 10 or more, 100 or more, 1000 or more, or 5000 or more) described in Table 13 (e.g., SEQ ID NOs:586-8317).
Yet other embodiments provide a method of determining the differentiation status of pluripotent or somatic cells, comprising: a) assaying the cells for the expression of one or more transcription factors or lincRNAs selected from those in FIGS. 3 and 6 and Tables 3 and 4; and b) determining the differentiation status of the cells based on the expression. In some embodiments, the presence or increased level of the cell transcription factors in FIG. 3 or Table 3 are indicative of cells that retain pluripotency. In some embodiments, the cell transcription factors are not Nanog, Sox2, Klf4, or Oct4. In some embodiments, the cell transcription factors selected from, for example, Mixip, Etv2, Zc3h11a, Zfp36, Isl2, Tfeb, Fig1a, Hsf2, or Hoxc11 are indicative of cells that retain pluiripotency. In some embodiments, the presence or increased level of the cell transcription factors shown in FIG. 6 or Table 4 is indicative of cells that have differentiated into neuronal cells. In some embodiments, the cell differentiation factors are not Neurog1, Brn2, or KIlf12. In some embodiments, the cell differentiation factors are selected from, for example, Ezh2, Suz12, or Jun.
Still further embodiments provide a method of differentiating pluripotent or somatic (e.g., fibroblast) cells into neuronal cells, comprising: inducing expression of one or more cell regulation factors shown in FIG. 6 or Table 4 in the pluripotent cells. In some embodiments, the cell differentiation factors are selected from, for example, Ezb2, Ngn1, Suz12, or Jun. In some embodiments, the inducing expression comprises contacting the pluripotent cells with a nucleic acid encoding one or more of the cell differentiation factors, contacting the pluripotent cells with an sgRNA that induces expression of one or more of the cell differentiation factors, or contacting the pluripotent cells with a small molecule that induces expression of the cell differentiation factors. In some embodiments, the method further comprises the step of determining the presence of increased level of expression of the cell differentiation factors shown in FIG. 6 or Table 4. In some embodiments, the presence or increased level of the cell differentiation factors shown in FIG. 6 or Table 4 is indicative of cells that have differentiated into neuronal cells.
Certain embodiments provide differentiated cells generated by the methods described herein.
Embodiments of the present disclosure provide a plurality of neuronal cells that express one or more cell differentiation regulation factors shown in FIG. 6 or Table 4 (e.g., one or more of Ezh2, Suz12 or Jun).
Further embodiments provide a method of inducing pluripotency or maintaining pluripotency of a cell line (e.g., a somatic or pluripotent cell line), comprising: inducing expression of one or more cell regulation factors shown in FIG. 3 or Table 3 in said cells (e.g., one or more of Mlxip, Etv2, Zinc Zc3h11a, Zfp36, Isl2, Tfeb, Fig1a, Hsf2, or Hoxc11).
Still other embodiments provide a plurality of pluripotent cells generated or maintained by the methods described herein.
In other embodiments, the present disclosure provides a plurality of pluripotent or iPSCs cells that express one or more cell regulation factors shown in FIG. 3 or Table 3 (e.g., one or more of Mlxip, Etv2, Zinc Zc3h11a, Zfp36, is12, Tfeb, Fig1a, Hsf2, or Hoxc11).
Some embodiments provide a method of transplanting cells, comprising: transplanting differentiated cells generated by the methods described herein into a subject in need thereof (e.g., a subject diagnosed with a disease or condition).
Further embodiments are described herein.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A-D shows that enhanced CRISPR activation mouse ES (CamES) cells allow efficient single sgRNA-directed gene activation and stem cell fate control. (A) Engineered eCRISPRa system in mouse ES cells for single sgRNA-mediated self-renewal and differentiation control. (B) A panel of sgRNAs tiling along the upstream regulatory region of Asc11 relative to transcription start site (TSS) in CamES cells show a gradient of efficient gene activation. (C) Effective neural (day 8) and muscle (day 12) differentiation of CamES cells using a single sgRNA to activate endogenous genes. (D) Time-course measurement of endogenous gene expression (Asc11, Brn2, Tuj1, and Map2) during differentiation for CamES cells −sgRNA, +negative control sgRNA, or +sgAsc11, and E14 mouse ES cells +Asc11 cDNA.
FIG. 2A-E shows the use of an sgRNA library to screen genes that maintain pluripotency and self-renewal in mouse ES cells. (A) Schematic representation of CRISPRa-mediated gain-of-function screening (dropout) of genes that maintain pluripotency and self-renewal in CamES cells using a sgRNA library. (B) Flow cytometry data of library-transduced CamES cells during serial passages and after SSEA1 sorting. Negative control, isotype antibody control. (C) Microscopic images showing bright Feld (BF), Oct4 staining, and DAPI of library-transduced CamES cells in −2i medium at passage 2, passage 10 before SSEA1 sorting and passage 10 after sorting. Scale bar, 100 μm. (D) Boxplot of normalized sgRNA counts for the plasmid library, library-transduced CamES cells at D0, and library-transduced CamES cells after SSEA1 sorting. (E) Detected sgRNA counts (sgRNAs with at least one count) in the plasmid library, library-transduced CamES cells at D0, and library-transduced CamES cells after SSEA1 sorting.
FIG. 3A-C shows validation of top hits from the CRISPRa self-renewal screen. (A) A scatter plot showing enrichment of sgRNAs for ranked top hit genes. (B) Fold change of mRNA expression measure by quantitative PCR for each gene using their individual sgRNA in CamES cells. (C) Microscopic images and flow cytometry analysis of pluripotency markers Oct4, Nanog, and SSEA1 in CamES cells transduced with 18 individual sgRNAs in −2i medium after 10 passages.
FIG. 4A-D shows functional characterization and deep sequencing analysis of sgMlxip-transduced CamES cells confirm maintenance of pluripotency in −2i medium. (A) Spontaneous differentiation of sgMlxip- or sgKlf2-transduced CamES cells after 10 passages shows generation of three germ layers. (B) RNA-seq paired scatter plot analysis of the Wnt pathway gene expression comparing CamES +sgMlxip in −2i medium with CamES +2i medium (left, R2=0.81), and comparing CamES −sgMlxip in −2i medium and CamES −2i medium at day 7 (right, R2=0.35). (C) RNA-seq scatter plot analysis of the MAPK pathway gene expression comparing CamES +sgMlxip in −2i medium with CamES +2i medium (left, R2=0.90), and comparing CamES +sgMlxip in −2i medium and CamES −2i medium at day 7 (right, R2=0.59). (D) Normalized mRNA expression for genes in the PI3K pathway for CamES +sgMlxip in −2i medium, CamES +2i medium, and CamES −2i medium at day 7.
FIG. 5A-D shows the use of sgRNA library to screen genes that promote neural differentiation of mouse ES cells. (A) Schematic representation of CRISPRa-mediated gain-of-function screening (non-dropout) of genes that promote neural differentiation in CamES cells using an sgRNA library. (B) Quantification by qPCR for neural marker Tuj1 and Map2 expression before and after MACS sorting. (C) Boxplot of normalized sgRNA counts for the plasmid library, sorted Tuj1-hCD8+ cells, and sorted Tuj1-hCD8− cells. (D) Detected sgRNA counts (sgRNAs with at least one count) in the plasmid library, sorted Tuj1-hCD8+ cells, and sorted Tuj1-hCD8− cells.
FIG. 6A-F shows validation of top hits from CRISPRa neural differentiation screen. (A) Scatter plot of sgRNA enrichment for ranked top hit genes. Only sgRNAs enriched in both replicates are shown. 20 genes and their most enriched sgRNAs (orange) are chosen for validation. (B) Fold change of mRNA expression measure by quantitative PCR for each gene using their individual sgRNA in Tuj1-hCD8 CamES cells. (C) Quantification of hCD8+ cells measured by flow cytometry in Tuj1-hCD8 CamES cells transduced without sgRNA, with 6 individual non-targeting sgRNAs, and with 20 individual sgRNA hits after 12-day differentiation. (D) Quantification of NCAM+ cells measured by flow cytometry in CamES cells transduced without sgRNA, with 6 individual non-targeting sgRNAs, and with 20 individual sgRNA hits after 12-day differentiation. (E) Microscopic images ofMap2 staining in Tuj1-hCD8 CamES cells transduced with individual sgRNAs after 12-day differentiation. Scale bar, 100 μm. (F) Characterization of staining various neural lineage markers (Tuj1, Map2, NeuN, Olig2, GFAP, and vGluT1) in Tuj1-hCD8 CamES cells transduced with individual sgRNAs after 12-day differentiation.
FIG. 7A-G shows functional characterization and deep sequencing analysis of sgJun-mediated CamES neural differentiation. (A) Representative traces of membrane potentials of differentiated neurons from Tuj1-hCD8 CamES cells transduced with sgJun in response to step-voltage (left) and step-current injections (right). (B) Principle component analysis of RNA-seq samples from D0, D2, D5, and D12 of sgJun-transduced CamES cells. (C) RNA-seq analysis showing time-course expression of 6 pluripotency genes and 6 neural lineage genes during differentiation of sgJun-transduced CamES cells. Error bars, s.d.±the mean of four biological replicates. (D) Gene ontology analysis of genes that are enriched in D5 and D12 differentiated neural cells (left, D5 versus D0; right, D12 versus D0). (E) Western blot showing protein expression of Jun and phosphorylated Jun at different time points during differentiation. P-Jun: phosphorylated Jun. (F) RNA-seq paired scatter plot analysis of the downstream genes targeted by the AP-1 complex formed between Jun and c-Fos. Left, D2 versus D0 (p=0.2); middle, D5 versus D0 (p=0.002); and right, D12 versus D0 (p=0.004). (G) Gaussian kernel density plot of expression of the Wnt pathway genes in sgJun-directed differentiated cells at different time points during the neural differentiation.
FIG. 8A-H shows generation of eCRISPRa by systematic optimization of the CRISPRa-SunTag system. (A) A multiple lentiviral eCRISPRa system. (B) Comparison of endogenous Brn2 activation efficiency using 12 individual sgRNAs targeting Brn2 or their mixture for the SFFV-driven scFv-stGFP-VP64 CRISPRa system. (C) Comparison of endogenous Brn2 activation efficiency for different promoters driving scFv-sfGFP-VP64. Data is normalized to the −sgRNA sample. (D) Comparison of endogenous Brn2 activation efficiency for 6 clonal cell lines each generated from EF1a- or PGK-driven scFv-sfGFP-VP64 systems. Data is normalized to the −sgRNA sample. (E) Comparison of endogenous Brn2 activation efficiency for 28 clonal cell lines generated from the PGK-driven scFv-sfGFP-VP64 system. (F) Characterization of CamES cells for the morphology, expression of pluripotency marker Oct4, and expression of eCRISPRa components. (G) Negative staining of Tuj1 (red) in CamES cells, CamES cells +sgControl, and E14 mouse ES cells +sgAsc11 after 12-day differentiation. DAPI is shown in blue. (H) Microscopic images showing cell morphology of CamES cells +sgAsc11 (top) and E14 mouse ES cells +Asc11 cDNA (bottom) at D0, D6, and D12 during differentiation.
FIG. 9A-B shows an experimental procedure and characterization of the CRISPRa self-renewal screen in mouse ES cells. (A) Time line scheme of the gain-of-function self-renewal screen using the sgRNA library. (B) Correlation of sequenced sgRNA counts in library-transduced CamES cells at D0 and after SSEA1 sorting.
FIG. 10 shows a ranked gene list based on the dropout self-renewal screen described in FIGS. 2 and 3.
FIG. 11A-C shows RNA sequencing and characterization of CamES cells +sgMlxip or +sgKlf2 cultured in −2i medium. (A) Heatmap illustrating mRNA expression of the pluripotency-associated genes and lineage specific genes for indicated samples. (B) Histogram plot showing distribution of ratios of the Wnt pathway gene expression for indicated samples. (C) mRNA expression of indicated MAPK pathway genes in CamES cells in −2i medium at D7, in +2i medium, and transduced with sgMlxip in −2i medium.
FIG. 12A-F shows an experimental procedure and characterization of CRISPRa gain-of-function neural differentiation screen. (A) Sequencing results of the Tuj1 locus in Tuj1-hCD8 CamES cells. (B) Flow cytometry data (right) showing the hCD8+ percentage of cells in sgAsc11-transduced Tuj1-hCD8 CamES cells after 8-day differentiation. (C) Comparison of Tuj1 and Map2 mRNA expression levels in differentiated cells with various initial seeding cell densities. (D) Quantification of Tuj1 and Map2 mRNA expression levels in CamES cells, CamES cells +sgControl, and CamES cells +sgLibrary during differentiation. (E) Staining of neural markers Tuj1 and Map2 in library-transduced Tuj1-hCD8 CamES cells. (F) Time line scheme of the gain-of-function neural differentiation screen using the sgRNA library in Tuj1-hCD8 CamES cells.
FIG. 13 shows a ranked gene list based on non-dropout neural differentiation screen shown in FIGS. 5 and 6.
FIG. 14A-F shows characterization of sgJun-directed neural differentiated cells and analysis of dropout and non-dropout screens. (A) Heatmap illustrating mRNA expression of representative pluripotency-associated, progenitor neural lineage, terminal neural lineage, endoderm lineage, and mesoderm lineage genes. (B) Time-course of normalized RNA-seq mRNA counts of 12 genes in the MAPK and Wnt pathways during sgJun-direct CamES cells differentiation. (C) A hypothesized model for endogenous Jun activation-induced neural differentiation by sgJun. (D) Toy example of dropout (left) and non-dropout screens (right). In dropout screens, negative cells drop out of the population and have little noticeable effect. (E) The percentage of screen hits in common with Tuj1-hCD8+/D0 for the Tuj1-hCD8−/D0. SSEA1+/D0, and Tuj1-hCD8+/Tuj1-hCD8− gene ranks at a given hit cutoff. (F) The top ten enriched genes as calculated for Tuj1-hCD8+ relative to day 0, Tuj1-hCD8− relative to day 0, and Tuj1-hCD8+ relative to Tuj1-hCD8-.
FIG. 15A-G shows a CRISPRi experimental screening platform for studying genetic interactions. (A) The experimental setup of the single and double CRISPRi screening platform for GI studies. (B-E) Characterization of biological replicates for single and double sgRNA libraries (R1—biological replicate 1; R2, biological replicate 2): (B) single library without Dox at day 20; (C) single library with Dox at day 20; (D) double library without Dox at day 16; (E) double library with Dox at day 16. (F) Comparison of single library with and without Dox at day 20. (G) Comparison of double library with and without Dox at day 16.
FIG. 16A-F shows a time-course comparison of sgRNA enrichment for single and double libraries and validation of sgRNA pairs for epistatic interactions. (A) Comparing day 0 sample to other time points (grey—day 3; red—day 7; blue—day 13) in the presence of Dox for the single library. (B) The 20 genes among 112 epigenetic factor genes that showed consistent depletion over time due to CRISPRi inhibition. (C) Comparing day 0 sample to other time points (grey—day 8; blue—day 16) in the presence of Dox for the double library. For the comparison without Dox, refer to Fig. S4B. (D) A selected combinations that showed consistent depletion over time due to multiplexed CRISPRi inhibition. (E-F) Validation of two pairwise sgRNAs (MRGBP & MED6; BRD7 & LEO1) for their combinatorial effects in suppressing cell growth and endogenous gene expression.
FIG. 17 shows a module map of chromatin-related genes based on a curated set of protein complexes.
FIG. 18A-E shows (A) Schematic representation of CRISPRa-mediated gain-of-function screenings that promote neuronal differentiation in CamES cells using an sgRNA library. (B) Frequency histograms of the top 3 enriched sgRNAs targeting genes indicated. (C) Quantification of PSA-NCAM+ cells were measured by flow cytometry in CamES cells transduced with three individual sgRNAs of each gene after 12-day differentiation. (D) Microscopic images of Map2 staining in CamES cells transduced with individual sgRNAs after 12-day differentiation. Scale bar, 100 μm. (E) Staining of various neuronal lineage markers (NeuN, Olig2, GFAP, GABA, and vGluT1) in CamES cells transduced with individual sgRNAs after 12-day differentiation.
FIG. 19A-F shows (A) Schematic representation of CRISPRa-mediated gain-of-function double screenings that promote neuronal differentiation in CamES cells using a double sgRNA library. (B) Schematic of the two-guide vector. (C) Reproducibility between the two replicates of the paired CRISPR screen of gene-targeting and negative control (or vice versa) guide pairs, mean±s.e.m. (standard error of the mean). (D) Interaction scores for a pair were computed by subtracting off the maximum of the guide-level effect sizes. (E) Interaction forming capacities of the two sgRNAs inducing different gene activation levels. (F) Quantification of PSA-NCAM+ cells measured by flow cytometry in CamES cells transduced with one single sgRNA or double sgRNAs after 12-day differentiation. Error bars represent standard deviation of three independent experiments.
FIG. 20A-E shows (A) Quantification of MAP2+ cells from MEFs infected with different gene combinations. Averages from 20 randomly selected visual fields are shown. Error bars indicate±s.d. (B) Representative images of Tuj1 staining of MEFs infected with different genes or gene combinations. Scale bar, 100 μm. (C) Ngn1 and Ezh2 induced MEF neuron cells express MAP2, Tuj1 and NeuN, synapsin, and GABA 14 days after infection. Scale bar, 100 μm. (D) Bar graph showing the percentage of Tbr1-positive neurons (Tbr1+) and GABA-positive neurons (GABA+) out of total neurons. (E) Ngn1 and Ezh2 induced perinatal TTF neuron cells express MAP2, Tuj1 and NeuN, synapsin, and GABA 26 days after infection. Scale bar, 100 μm.
FIG. 21A-I shows that MEF-derived induced neurons show functional synaptic properties. (A) Recording electrode patched onto a sfGFP-positive cell with a stimulation electrode (middle panel). The right panel is a merged picture of BF and fluorescence images showing that the recorded cell is sfGFP-positive. (B) Representative traces of whole-cell currents in voltage-clamp mode; cells were held at −80 mV. Step depolarization from 70 mV to +40 mV at 10-mV intervals was delivered (lower panel). (C) Representative trace of evoked membrane potential by +40 pA current injection (lower panel) in current-clamp mode held at −75 mV. Application of 100 nM tetrodotoxin (TTX), a selective blocker of voltage-gated sodium channels, inhibited the action potential. (D) Inward sodium currents were evoked from an induced neurons, and application of 500 nM TTX inhibited these currents. Step depolarization from −70 mV to +60 mV at 10-mV intervals was delivered; cells were held at −80 mV (right panel); a presentative trace of whole-cell current with and without TTX at −10 mV membrane potential in voltage-clamp mode is shown (left panel). (E) Outward potassium currents were evoked from an induced neurons, and application of 5 mM tetraethylammonium (TEA) inhibited these currents. Step depolarization from −70 mV to +60 mV at 10-mV intervals was delivered; cells were held at −80 mV (right panel); a presentative trace of whole-cell current with and without TEA at +60 mV membrane potential in voltage-clamp mode is shown (left panel). (F) Spontaneous EPSCs were recorded from induced neurons. (G) Spontaneous action potentials recorded from an induced neuron (left panel). Application of 100 nM TTX blocked the action potentials (middle panel). Washout of TTX reversed the blockade (right panel). (1-) Representative traces of evoked excitatory spontaneous postsynaptic currents (EPSCs) recorded from an induced neuron (left panel). Application of 30 μM DNQX (6,7-dinitroquinoxaline-2,3-dione), an AMPA/kainate glutamate receptor antagonist, blocked the response of EPSCs (middle panel). Washout of DNQX reversed the blockade (right panel). (1) Representative traces of evoked EPSCs recorded from an induced neuron (left panel). Application of 30 μM BIC (Bicuculline), a GABA receptor antagonist, slightly increased the frequency and amplitude of EPSCs (middle panel). Washout of BIC reversed the increase (right panel). F, and H-I, Cells were recorded at a holding potential (Vh) of −60 mV. Error bars indicate±s.d. of cell counts. Scale bar, 10 μm.
FIG. 22A-E shows generation of the CRISPRa and CRISPRa knock-in cell lines. (A) A multiple lentiviral CRISPRa system. (B) Characterization of CamES cells for the morphology, expression of pluripotency marker Oct4, and expression of CRISPRa components. Scale bars, 100 μm. (C) Schematic of the clonal CamES cell line carrying a biallelic IRES-hCD8 insertion at the Tuj1 locus. (D) Sequencing results of the Tuj1 locus in Tuj1-hCD8 CamES cells. (E) Quantification by qPCR for neuronal markers Tuj1 and Map2 expression before and after MACS sorting.
FIG. 23A-G shows (A) Time line scheme of the neural differentiation screens using the sgRNA library in Tuj1-hCDS CamES cells. (B) Quantification of Tuj1 and Map2 mRNA expression levels in CamES cells, CamES cells +sgControl, and CamES cells +sgLibrary during differentiation. Error bars, s.d.±the mean of three independent experiments. (C) Staining of neural markers Tuj1 and Map2 in library-transduced Tuj1-hCD8 CamES cells. Scale bar, 100 μm. (D) Boxplot. of normalized sgRNA counts for the plasmid library, sorted Tuj1-hCD8+ cells, and sorted Tuj1-hCD8− cells. (E) The top ten enriched genes as calculated for Tuj1-hCD8+ relative to day 0, Tuj1-hCD8− relative to day 0, and Tuj1-hCD8+ relative to Tuj1-hCD8−. (F) Toy example of sgRNA stochastic representation in the screening system. (G) The percentage of screen hits in common with Tuj1-hCDS+/D0 for the Tuj1-hCD8−/D0, SSEA1+/D0, and Tuj1-hCD8+/Tuj1-hCD8− gene ranks at a given hit cutoff.
FIG. 24A-D shows (A) Quantification of PSA-NCAM+ cells were measured by flow cytometry in CamES cells transduced with three individual sgRNAs of each gene after 12-day differentiation. (B) Quantification of hCD8+ cells measured by flow cytometry in Tuj1-hCD8 CamES cells transduced without sgRNA, with 6 individual non-targeting sgRNAs, and with 19 individual sgRNAs after 12-day differentiation. Error bars represent standard deviation of three independent experiments. (C) Quantification of PSA-NCAM+ cells were measured at day 10 by flow cytometry in E14 cells after induction of different transgenes or negative control transgene BFP. Error bars represent standard deviation of three independent experiments. (D) Staining of various neuronal lineage markers (Tuj1, NeuN, Olig2, GFAP, GABA, and vGluT1) in CamES cells transduced with individual sgRNAs after 12-day differentiation. Scale bar, 100 μm.
FIG. 25 shows quantification of MAP2+ cells from MEFs infected with different genes.
FIG. 26A-E shows (A) The distribution of guides for the top 19 hits in green against an equal number of randomly selected negative control guides. (B) Variable gene effects and mixing proportions. (C) The estimated gene effect sizes plotted versus the estimated gene specific mixing proportions. (D) The estimated feature coefficients and their 80% credible interval from the model described in Example 4. (E) The distribution of average log 2 fold change of guides in the corresponding feature (top).
FIG. 27A-D shows (A) Cloning strategy for final two-guides vector. (B) Sequencing strategy to analyze the sgRNA sequences for the double sgRNA library. (C) Empirical Bayes fit of the null distribution of the constructed test statistic using the R package locfdr. (D) Correlation of sequenced sgRNA counts in library-transduced CamES cells at D0, Tuj1-hCD8+ cells and Tuj1-hCD8− cells after hCD8 sorting.
FIG. 28A-1H shows (A) Ngn1 and Foxo1 induced MEF neuron cells express MAP2, Tuj1 and NeuN, synapsin, and GABA 14 days after infection. Scale bar, 100 μm. (B) Bar graph showing the percentage of Tbr1-positive neurons (Tbr1+) and GABA-positive neurons (GABA+) out of total neurons. (C) Ngn1 and Foxo1 induced perinatal TTF neuron cells express MAP2, Tuj1, and NeuN, synapsin, and GABA 26 days after infection. Scale bar, 100 μm. (D) Inward sodium currents were evoked from induced neurons, and application of 500 nM TTX inhibited these currents. (E) Outward potassium currents were evoked from an induced neurons, and application of 5 mM tetraethylammonium (TEA) inhibited these currents. (F) Spontaneous action potentials recorded from an induced neuron (left panel). Application of 100 nM TTX blocked the action potentials (middle panel). Washout of TTX reversed the blockade (right panel). ((G) Representative traces of evoked excitatory spontaneous postsynaptic currents (EPSCs) recorded from an induced neuron (left panel). Application of 30 μM DNQX (6,7-dinitroquinoxaline-2,3-dione), an AMPA/kainate glutamate receptor antagonist, blocked the response of EPSCs (middle panel). Washout of DNQX reversed the blockade (right panel). (H) Representative traces of evoked EPSCs recorded from an induced neuron (left panel). Application of 30 μM BIC (Bicuculline), a GABA receptor antagonist, slightly increased the frequency and amplitude of EPSCs (middle panel). Washout of BIC reversed the increase (right panel). G and H, Cells were recorded at a holding potential (Vh) of −60 mV. Error bars indicate±s.d. of cell counts.
FIG. 29 shows representative images of Tuj1 staining of MEFs infected with different gene combinations. Scale bar, 100 μm.
DEFINITIONS As used herein the term “stem cell” (“SC”) refers to cells that can self-renew and differentiate into multiple lineages. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferates into the tissue's mature, fully formed cells. Stem cells may be derived, for example, from embryonic sources (“embryonic stem cells”) or derived from adult sources. For example, U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT publications WO 00/52145 and WO 01/00650 (herein incorporated by reference in their entireties) describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines.
Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells).
As used herein, the term “totipotent cell” refers to a cell that is able to form a complete embryo (e.g., a blastocyst).
As used herein, the term “pluripotent cell” or “pluripotent stem cell” refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types. A pluripotent cell can be self-renewing, and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form a new blastocyst.
As used herein, the term “induced pluripotent stem cells” (“iPSCs”) refers to a stem cell induced from a somatic cell, e.g., a differentiated somatic cell, and that has a higher potency than said somatic cell. iPS cells are capable of self-renewal and differentiation into mature cells.
As used herein, the term “multipotent cell” refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.
As used herein, the term “progenitor cell” refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.
As used herein, the term “embryonic stem cell” (“ES cell” or ESC”) refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5-day-old human embryo), and has the ability to yield many or all of the cell types present in a mature animal.
As used herein the term “feeder cells” refers to cells used as a growth support in some tissue culture systems. Feeder cells may, for example, embryonic striatum cells or stromal cells.
As used herein, the term “chemically defined media” refers to culture media of known or essentially-known chemical composition, both quantitatively and qualitatively. Chemically defined media is free of all animal products, including serum or serum-derived components (e.g., albumin).
DETAILED DESCRIPTION Provided herein are compositions and methods for identifying and using stem cell differentiation regulation factors. For example, in some embodiments, provided herein are compositions and methods for identifying stem cell differentiation regulation factors using marker gene expression libraries. Also provided herein are compositions and methods for generating differentiated and induced cells lines and uses of such cell lines.
The RNA-guided microbial endonuclease CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR associated protein 9) system was recently repurposed as a tool for sequence-specific gene editing and transcriptional regulation (Cho et al., 2013 Nat. Biotechnol. 31, 230-232; Cong et al., 2013 Science 339, 819-823; Fu et al., 2014 Nat. Biotechnol. 32, 279-284; Jinek et al. Science 337, 816-821, 2012; Mali et al., 2013b Science 339, 823-826; Qi et al., 2013 Cell 152, 1173-1183; Ran et al., 2015 Nature 520, 186-191; Yu et al., 2015 Cell Stem Cell 16, 142-147). The nuclease-dead Cas9 (dCas9) fused with transcription activator domains allows endogenous genes activation, leading to CRISPR activation (CRISPRa) methods (Chavez et al., 2015 Nat. Method. 12, 326-328; Cheng et al., 2013 Cell Res. 23, 1163-1171; Gilbert et al., 2013 Cell 154, 442-451; Hilton et al., 2015 Nat. Biotechnol. 33, 510-517; Konermann et al., 2015 Nature 517, 583-588; Maeder et al., 2013 Nat. Method. 10, 977-979; Mali et al., 2013a Nat. Biotechnol. 31, 833-838; Perez-Pinera et al., 2013 Nat. Method. 10, 973-976; Tanenbaum et al., 2014 Cell 159, 635-646; Zalatan et al., 2015 Cell 160, 339-350). Previous work demonstrated that CRISPR activation of endogenous genes allowed, in principle, somatic cell reprogramming and directed cell differentiation (Black et al., 2016 Cell Stem Cell 19, 406-414; Chakraborty et al., 2014 Stem Cell Reports 3, 940-947; Chavez et al., 2015 Nat. Method. 12, 326-328; Wei et al., 2016 Sci. Rep. 6, 19648). However, since these studies relied on using a mixture of multiple sgRNAs for activating a single gene and inducing differentiation, applying these methods for large-scale activation screening has been a major challenge.
Unlike cell growth phenotypes that entail a dropout live-or-dead process, cell fate determination is a dynamic, stochastic process that often generates a heterogeneous cell population with diverse phenotypes (e.g., non-dropout) (Hanna et al., 2009 Nature 462, 595-601; Johnston and Desplan, 2010 Annu. Rev. Cell Dev. Biol. 26, 689-719). This imposes another challenge to simply perform dropout screens that distinguish lineage specification processes from spontaneous differentiation events. Furthermore, because developmental programs are highly dependent on the expression level of endogenous genes (Niwa et al., 2000 Nat. Genet. 24, 372-376; Papapetrou et al., 2009 Proc. Natl. Acad. Sci. USA 106, 12759-12764), gain-of-function screens that allow very efficient gene activation (comparable to cDNA overexpression) while covering a broad range of expression offer more promise for identifying candidate genes driving cell lineages. To date, two reports used CRISPRa for cell growth-based dropout screens (Gilbert et al., 2014 Cell 159, 647-661; Konermann et al., 2015 Nature 517, 583-588). However, the application of CRISPRa screens for the systematic inference of cell fate determination has not yet been established.
Experiments described herein overcame these challenges by developing a CRISPR activation (CRISPRa)-mediated gain-of-function screening approach to identify transcription factors (TFs) important for stem cell fate determination. An enhanced CRISPRa system was developed in mouse embryonic stem (ES) cells that efficiently activates endogenous genes and drives cell lineage differentiation. A single sgRNA was sufficient to induce neuron or muscle differentiation. Based on the system, a large-scale sgRNA library (>50,000 sgRNA) was used to target all putative endogenous TF genes (˜800) and a small set of noncoding RNA genes (50). Targeting a single gene using multiple sgRNAs (>60 sgRNA per gene) allowed activating each gene to a broad range of expression levels. A CRISPRa dropout screen was used to identify genes that promote stem cell self-renewal, as well as a non-dropout screen for inducing neural differentiation. The top gene hits were validated using individual sgRNAs, and it was observed that all hits could maintain self-renewal. For neural differentiation, it was confirmed that 19 out of top 20 gene hits could induce efficient neural differentiation. For both screens, the lists of gene hits include known TF factors and those TFs and noncoding RNAs that are not previously related to self-renewal maintenance or neural differentiation. Different identified TFs preferentially induced different types of neurons. Deep sequencing and functional analysis of a few gene hits (Mlxip for self-renewal and Jun for neural differentiation) confirmed their functions for driving desired cellular processes.
Thus, the compositions and methods provide herein allow for the identification of the relevant factors necessary, sufficient, and/or useful for controlling differentiation of stem cells into any desired fat. The transcription factors identified herein and identifiable using the compositions and methods described herein provide target and reagents for differentiation of cells an provide the cells made therefrom that find use as research tools, drug screening targets, and therapeutics (e.g., via cell transplantation into a host).
The CRISPRa gain-of-function screens and stem cell libraries described herein find use in research, therapeutic, and screening applications to determine differentiation factors for a variety of stem cells. The differentiation factors identified further find use in stem cell differentiation for research, screening, and clinical applications.
1. Identification of Differentiation Factors As described herein, embodiments of the present disclosure provide compositions and methods for identifying stem cell differentiation regulation factors. In some embodiments, the methods utilize a modified pluripotent or multipotent (e.g., stem cell) line. The present disclosure is not limited to particular cell lines. Examples include, but are not limited iPSC, embryonic stem cells, adult stem cells, and the like.
In some embodiments, the CRISPR activation system comprises a dCas9 construct under the transcriptional control of a first promoter. In some embodiments, the dCas9 is fused to a peptide epitope. In some embodiments, the activation system further comprises a VP64 transactivation domain under the transcriptional control of a second promoter. In some embodiments, the VP64 transactivation domain is fused to a peptide that specifically binds to the peptide epitope. In some embodiments, the activation system further comprises a selection marker under the transcriptional control of a third promoter. In some embodiments, each of the first, second, and third promoters are different than each other.
In some embodiments, cell lines for determination of differentiation regulation factors are pluripotent cells modified with a dead Cas9/transactivator activation system. For example in some embodiments, cells comprise a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 is fused to a signal activation component (e.g., a plurality of peptide epitopes as described in Tanenbaum et al., (2014). Cell 159, 635-646; herein incorporated by reference in its entirety). In some embodiments, the cell lines further comprise a single chain variable chain antibody fragment specific for the peptide epitope fused to a tranactivator domain (e.g., VP64; See e.g., Beerli et al., Proc Natl Acad Sci USA. 1998 Dec 8; 95(25): 14628-14633; herein incorporated by reference in its entirety) and a transactivator polypeptide. In some embodiments, the activation components are provided on a vector (e.g., retroviral vector, adenoviral viral vector, adeno-associated vector, lentiviral vector, etc.). In some embodiments, cells further overexpress endogenous Brn2 (e.g., via an sgRNA that targets activation of Brn2).
In some embodiments, the cells lines are next contacted with a plurality of sgRNAs (e.g., targeting cell differentiation regulation factors). In some embodiments, sgRNAs target transcription factors or non-coding RNAs (e.g., lincRNAs). In some embodiments, more than one (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) sgRNAs specific for each differentiation factor are utilized. In some embodiments, sgRNAs are provided on vectors (e.g., retroviral vector, adenoviral viral vector, adeno-associated vector, lentiviral vector, etc.). In some embodiments, cells are further contacted with a plurality of non-targeting sgRNAs (e.g., to serve as negative controls). In some embodiments, a double CRISPR screen is performed using dual-sgRNA-constructs comprising two (or more) sgRNAs to screen for interactions between multiple cell differentiation factors in combination.
In some embodiments, the method further comprises contacting the cell differentiation factors with a fibroblast or other cell line and identifying cell differentiation factors that promote transdifferentiation of the fibroblast cell line. In some embodiments, the fibroblast cell line is contacted with combinations of two or more cell differentiation factors. In some embodiments, the cell differentiation factors that promote differentiation are combinations of Ngn1+Brn2, Ezh2+Brn2, Mecom+Ezh2, Ngn1+Ezh2, or Ngn1+Foxo1.
In some embodiments, the method comprises or further comprises the step of performing a CRISPR gene repression screen. For example, in some embodiments, the CRISPR repression screen comprises: a) contacting a pluripotent cell that expresses dCas9 fused to a transcription repressor domain (e.g., KRAB) with a plurality of sgRNAs specific for repression of a plurality of cell differentiation factors; b) sorting the library to identify cells that retain pluripotency or differentiate; and c) identifying cell differentiation factors that induce or prevent differentiation of said pluripotent cells. In some embodiments, the CRISPR repression screen and the CRISPR activation screen are performed in the same or different pluripotent cells. In some embodiments, the CRISPR repression screen and the CRISPR activation screen are performed simultaneously using vectors comprising a first sgRNA specific for activation of a first cell differentiation factor and a second sgRNA specific for repression of a second cell differentiation factor.
The resulting gene activation library from CRISPR activation and/or repressor cells are then further analyzed as described below. For example, in some embodiments, following delivery of sgRNAs, cells are cultured and cells that retain pluripotency or differentiate are identified. In some embodiments, cells are sorted based on the presence or absence of differentiation or pluiptency markers.
In some embodiments, in order to identify regulation factors for pluipotency, cells are cultured under conditions that do not inhibit differentiation (e.g., in media lacking inhibitors of GSK3 and ERK pathways). In some embodiments, pluripotent cells are sorted by identifying and selecting (e.g., using flow cytometry) cells that express SSEA1 after culture.
In some embodiments, cells that differentiate are identified by sorting for cells that express differentiation markers specific to the final cell type. For example, in some embodiments, cells that differentiate into neuronal cells are identified by sorting for cells that express Tuj1.
In some embodiments, cell differentiation factors are activated and analyzed in pairs or groups (e.g., as described in Example 2 below) in order to identify combined effects of between different factors.
In some embodiments, after selection, cell differentiation regulation factors are identified by identifying sgRNAs that persist in the sorted cells. In some embodiments, sequencing (e.g., deep sequencing) is used to identify sgRNAs. In some embodiments, sequencing methods further comprises comparing the level of said sgRNAs to the level of non-targeting sgRNAs.
In deep sequencing, a high number of replicates of each sequencing read (e.g., at least 10, 20, 30, 40, 50, or 100) are used to improve accuracy. The present disclosure is not limited to a particular sequencing technique. Exemplary sequencing techniques are described below. A variety of nucleic acid sequencing methods are contemplated for use in the methods of the present disclosure including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. Many of these sequencing methods are well known in the art. See, e.g., Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al., Proc. Natl. Acad. Sci. USA 74:560-564 (1977); Drmanac, et al., Nat. Biotechnol. 16:54-58 (1998); Kato, Int. J. Clin. Exp. Med. 2:193-202 (2009); Ronaghi et al., Anal. Biochem. 242:84-89 (1996); Margulies et al., Nature 437:376-380 (2005); Ruparel et al., Proc. Natl. Acad. Sci. USA 102:5932-5937 (2005), and Harris et al., Science 320:106-109 (2008); Levene et al., Science 299:682-686 (2003); Korlach et al., Proc. Natl. Acad. Sci. USA 105:1176-1181 (2008); Branton et al., Nat. Biotechnol. 26(10):1146-53 (2008); Eid et al., Science 323:133-138 (2009); each of which is herein incorporated by reference in its entirety.
Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g., Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated by reference in their entirety). NGS methods can be broadly divided into those that typically use template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos BioSciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/Ion Torrent, and Pacific Biosciences, respectively.
Other emerging single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S. patent application Ser. No. 11/781,166; each herein incorporated by reference in their entirety) in which immobilized, primed DNA template is subjected to strand extension using a fluorescently-modified polymerase and florescent acceptor molecules, resulting in detectible fluorescence resonance energy transfer (FRET) upon nucleotide addition.
Exemplary cell regulation factors indicative of cells that retain pluripotency or differentiate are described in the Figures and Tables herein. For example, in some embodiments, cell transcription factors that retain pluripotency are one or more of the regulation factors shown in FIG. 3 or Table 3 and cell differentiation factors that are associated with differentiation into neuronal cells are one or more of the regulation factors shown in FIG. 6 or Table 4.
The cell differentiation factors identified using the described methods find use in a variety of applications. Exemplary uses are described herein.
II. Cell Lines and Libraries and Uses Thereof In some embodiments, the present disclosure provides cells lines, kits, and systems for use in the described methods. For example, in some embodiments, provided herein are libraries of modified pluripotent cells as described above. For example, in some embodiments, the cells comprise a dCas9 construct under the transcriptional control of a first promoter. In some embodiments, the dCas9 is fused to a peptide epitope. In some embodiments, the cells comprise a VP64 transactivation domain under the transcriptional control of a second promoter. In some embodiments, the VP64 transactivation domain is fused to a peptide that specifically binds to the peptide epitope. In some embodiments, the cells comprise a selection marker under the transcriptional control of a third promoter. In some embodiments, each of the first, second, and third promoters are different than each other.
In some embodiments, cells express i) nuclease dead Cas9 fused to a plurality of peptide epitopes; ii) a single chain variable chain antibody fragment specific for said peptide epitope fused to a VP64 tranactivator domain; and iii) a transactivator polypeptide.
In some embodiments, the cell lines described herein find use in screening (e.g., drug screening) and research applications as described below.
In some embodiments, provided herein are kits and systems comprising the cell lines described herein. In some embodiments, kits and systems further comprise a plurality of sgRNAs specific for activation of pluripotent cell differentiation factors. In some embodiments, the kit or system comprises one or more sgRNAs (e.g., 10 or more, 100 or more, 1000 or more, or 5000 or more) described in Table 13 (e.g., SEQ ID NOs:586-8317).
In some embodiments, kits and systems further comprise reagents for analysis of one or more properties of the cell lines (e.g., pluripotency or differentiation), reagents for sequencing the cells to identify the presence of sgRNAs, reagents for further downstream analysis (e.g., molecular analysis, toxicity screening, drug screening, or cellular activity assays), or computer software and computer systems for analyzing data.
III. Differentiation Methods In some embodiments, the present disclosure provides compositions and methods for differentiating cells into multipotent or specific cell types. The present disclosure is not limited to particular target cell types. Examples include, but are not limited to, epithelial cells (e.g., exocrine secretory epithelial cells, hormone secreting cells (e.g., islet cells), keratinizing epithelial cells (e.g., skin cells), central nervous system cells (e.g., neuronal cells), blood cells, and organ cells.
In some embodiments, differentiation is induced by increasing expression of cellular regulation factors identified using the methods described herein. In some embodiments, expression is induced by exogenously introduced differentiation genes. In one embodiment, the exogenously introduced gene may be expressed from a chromosomal locus different from the endogenous chromosomal locus of the gene. Such chromosomal locus may be a locus with open chromatin structure, and contain gene(s) dispensible for a somatic cell. In other words, the desirable chromosomal locus contains gene(s) whose disruption will not cause cells to die. Exemplary chromosomal loci include, for example, the mouse ROSA 26 locus and type II collagen (Col2a1) locus (See Zambrowicz et al., 1997) The exogenously introduced pluripotency gene may be expressed from an inducible promoter such that their expression can be regulated as desired.
In some embodiments, the exogenously introduced gene is transiently transfected into cells, either individually or as part of a cDNA expression library. The cDNA library is prepared by conventional techniques. Briefly, mRNA is isolated from an organism of interest. An RNA-directed DNA polymerase is employed for first strand synthesis using the mRNA as template. Second strand synthesis is carried out using a DNA-directed DNA polymerase which results in the cDNA product. Following conventional processing to facilitate cloning of the cDNA, the cDNA is inserted into an expression vector such that the cDNA is operably linked to at least one regulatory sequence. The choice of expression vectors for use in connection with the cDNA library is not limited to a particular vector. Any expression vector suitable for use in mammalian cells is appropriate. In one embodiment, the promoter which drives expression from the cDNA expression construct is an inducible promoter. The term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel: Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express cDNAs. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
In some embodiments, the CRISPR activation and/or repression system is expressed from an inducible promoter. The term “inducible promoter”, as used herein, refers to a promoter that, in the absence of an inducer (such as a chemical and/or biological agent), does not direct expression, or directs low levels of expression of an operably linked gene (including cDNA), and, in response to an inducer, its ability to direct expression is enhanced, Exemplary inducible promoters include, for example, promoters that respond to heavy metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al. Nature (1982), 296, 39-42), to thermal shocks, to hormones (Lee et al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock et al. Nature (1987), 329, 734-736; Israel and Kaufman, Nucleic Acids Res. (1989), 17, 2589-2604), promoters that respond to chemical agents, such as glucose, lactose, galactose or antibiotic.
A tetracycline-inducible promoter is an example of an inducible promoter that responds to an antibiotics. See Gossen et al., 2003. The tetracycline-inducible promoter comprises a minimal promoter linked operably to one or more tetracycline operator(s). The presence of tetracycline or one of its analogues leads to the binding of a transcription activator to the tetracycline operator sequences, which activates the minimal promoter and hence the transcription of the associated cDNA and the expression of CRISPR activation and/or repression system. Tetracycline analogue includes any compound that displays structural homologies with tetracycline and is capable of activating a tetracycline-inducible promoter. Exemplary tetracycline analogues includes, for example, doxycycline, chlorotetracycline and anhydrotetracycline.
In some embodiments, expression of cell differentiation factors is induced via activating sgRNAs as described herein (e.g., Example 1). One or more sgRNAs are introduced into a pluripotent cell that expresses a CRISPR activation system (e.g., those described herein or other suitable system).
In some embodiments, differentiation is induced via small molecules that active expression or activity of cell differentiation genes or downstream signaling partners.
In some embodiments, cells are cultured under conditions that promote differentiation. In some embodiments, cultures are adherent cultures, e.g., the cells are attached to a substrate. The substrate is typically a surface in a culture vessel or another physical support, e.g. a culture dish, a flask, a bead or other carrier. In some embodiments, the substrate is coated to improve adhesion of the cells and suitable coatings include laminin, poly-lysine, poly-ornithine and gelatin. In some embodiments, the cells are grown in a monolayer culture or in suspension or as balls or clusters of cells. At higher densities, cells may begin to pile up on each other, but the cultures are essentially monolayers or begin as monolayers, attached to the substrate.
Cells differentiated using the methods described herein find use in a variety of research, screening, and clinical applications. In some embodiments, cells are used to prepare antibodies and cDNA libraries that am specific for the differentiated phenotype. General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.), Current Protocols in Immunology (Coligan et al., eds.); and Methods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags GmbH). General techniques involved in preparation of mRNA and cDNA libraries are described in RNA Methodologies: A Laboratory Guide for Isolation and Characterization (R. E. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogeneous cell populations are particularly suited for use in drug screening and therapeutic applications.
In some embodiments, the cells generated by methods provided herein or the above-described cell lines are used to screen for agents (e.g., small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the cells. Particular screening applications relate to the testing of pharmaceutical compounds in drug research. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change. Any suitable assays for detecting changes associated with test agents may find use in such embodiments. The screening may be done, for example, either because the compound is designed to have a pharmacological effect on specific cell types, because a compound designed to have effects elsewhere may have unintended side effects, or because the compound is part of a library screen for a desired effect. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects. In some applications, compounds are screened for cytotoxicity.
In some embodiments, methods and systems are provided for assessing the safety and efficacy of drugs that act upon the differentiated cells, or drugs that might be used for another purpose but may have unintended effects upon the cells. In some embodiments, cells described herein find use in high throughput screening (ITS) applications. In some embodiments, a HTS screening platform is provided (e.g., cells and plates) that allows for the rapid testing of large number (e.g., 1×103, 1×104, 1×105, 1×106 (or more) of agents (e.g., small molecule compounds, peptides, etc.).
In some embodiments cells generated using methods and reagents described herein are utilized for therapeutic delivery to a subject (e.g., a subject with a disease or other condition). Cells may be placed directly in contact with subject tissue or may be otherwise sealed or encapsulated (e.g., to avoid direct contact). In embodiments in which cells are encapsulated, exchange of factors, nutrients, gases, etc. between the encapsulated cells and the subject tissue is allowed. In some embodiments, cells are implanted/transplanted on a matrix or other delivery platform.
If appropriate, cells are co-administered with one or more pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells.
Support materials suitable for use for purposes of the present disclosure include tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures, which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present disclosure. See, for example, the materials disclosed in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830, 6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S. Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264 and 6,333,029.
Cells generated with methods and reagents herein may be implanted as dispersed cells or formed into implantable clusters. In some embodiments, cells are provided in biocompatible degradable polymeric supports; porous, permeable, or semi-permeable non-degradable devices; or encapsulated (e.g., to protect implanted cells from host immune response, etc.). Cells may be implanted into an appropriate site in a recipient. Suitable implantation sites depend on the cell type and may include, for example, the brain, spinal cord, skin, liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach, or a subcutaneous pocket.
In some embodiments, cells or cell clusters are encapsulated for transplantation into a subject. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles, and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, herein incorporated by reference in its entirety).
Methods of preparing microcapsules include those disclosed by Lu M Z, et al. Biotechnol Bioeng. 2000, 70: 479-83; Chang T M and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu M Z, et al., J. Microencapsul. 2000, 17: 245-51; herein incorporated by reference in their entireties. For example, microcapsules may be prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; herein incorporated by reference in its entirety). In some embodiments, microcapsules are based on alginate, a marine polysaccharide (Sambanis. Diabetes Technol. Ther. 2003, 5: 665-8; herein incorporated by reference in its entirety) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
In some embodiments, cells generated using methods and reagents described herein are microencapsulated for transplantation into a subject (e.g., to prevent immune destruction of the cells). Microencapsulation of cells provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs). In some embodiments, cells and/or cell clusters are microencapsulated in a polymeric, hydrogel, or other suitable material, including but not limited to: poly(orthoesters), poly(anhydrides), poly(phosphoesters), poly(phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly(L-lysine), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic acid-co-lysine), poly(lactic acid-graft-lysine), polyanhydrides, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly(anhydride-co-imides), poly(amides), poly(ortho esters), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate), poly(caprolactone), poly(carbonates), poly(amino acids), poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes), poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, polystyrene, polysaccharides, alginate, hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc., and their derivatives, co-polymers, and mixtures thereof. In some embodiments, cells are microencapsulated in an encapsulant comprising or consisting of alginate. Cells may be embedded in a material or within a particle (e.g., nanoparticle, microparticle, etc.) or other structure (e.g., matrix, nanotube, vesicle, globule, etc.). In some embodiments, microencapsulating structures are modified with immune-modulating or immunosuppressive compounds to reduce or prevent immune response to encapsulated cells. For example, in some embodiments, cells are encapsulated within an encapsulant material (e.g., alginate hydrogel) that has been modified by attachment of an immune-modulating agent (e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1). In some embodiments, such an immune modulating agent is a T-cell chemorepellent and/or a pro-survival factor.
In some embodiments, cells generated using methods and reagents described herein are macroencapsulated for transplantation into a subject. Macroencapsulation of cells, for example, within a permeable or semi-permeable chamber, provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs), prevents spread of cells to other tissues or areas of the body, and/or allows for efficient removal of cells. Suitable devices for macroencapsulation include those described in, for example, U.S. Pat. No. 5,914,262; Uludag, et al., Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, herein incorporated by reference in their entireties.
Other encapsulation (micro or macro) devices and methods may find use in embodiments described herein. For example, methods and devices described in U.S. Pub No. 20130209421, U.S. Pat. No. 8,785,185, each of which are herein incorporated by reference in their entireties, are within the scope of embodiments described herein.
IV. Differentiation Factors As described above and in the examples below, a number of new transcription factor and other regulatory factors involved in the regulating the differentiation processes have been discover using the screening methods described herein. These factors find use in generating stem cells or differentiated cells have desired properties for use in research, drug screening, and therapeutic applications.
In some embodiments, individual or combinations of these factors are used to induce differentiation in a stem cell to obtain differentiated cells or multipotent cells of a particular lineage (e.g., neural stem cells). In some embodiments, such factor are introduced exogenously to stem cells in vitro or in vivo (e.g., via expression vector, etc). In some embodiments, endogenous factors are up or down regulated by providing activators or inhibitors of endogenous expression.
In some embodiments, individual or combinations of these factors are used to induce differentiation in a somatic cell (e.g., fibroblast, neuronal cell, etc).
In some embodiments, individual or combinations of these factors are used to maintain or induce pluripotency in a cell line. In some embodiments, such factor are introduced exogenously to stem cells or somatic cells in vitro or in vivo (e.g., via expression vector, etc). In some embodiments, endogenous factors are up or down regulated by providing activators or inhibitors of endogenous expression.
In some embodiments, one or more of the markers described in Tables 3 and 4 are targeted. In some embodiments, provided herein are one or more sgRNAs (e.g., 10 or more, 100 or more, 1000 or more, or 5000 or more) described in Table 13 (e.g., SEQ ID NOs:586-8317) for use in targeting the described markers.
In some embodiments, provided herein are cell generated by such methods and the use of such cells, for example, in drug screening, diagnostic, and therapeutic indications.
Where transcription factors are introduced as peptides, in some embodiments they are complexed with cell membrane permeable peptides (e.g., Tat protein, penetratin, etc.) to facilitate entry into target cells.
EXPERIMENTAL Example 1 CRISPR Activation Screens Identify Genes Promoting Self-Renewal and Neuronal Differentiation of Stem Cells Methods sgRNA Library Construction
The oligo library was PCR amplified, gel purified and ligated to the linearized backbone vector (pSLQ1373) digested with BstXI and BlpI using In-Fusion cloning (Clontech).
Cell Culture
E14 mouse ES cells and CamES cells were maintained on gelatin coated tissue culture plates with basal medium (50% Neurobasal, 50% Dulbecco modified Eagle medium (DMEM) Ham's nutrient mixture F12, 0.5% NEAA, 0.5% Sodium Pyruvate, 0.5% GlutaMax, 0.5% N2, 1% B27, 0.1 mM β-mercaptoethanol and 0.05 g/L bovine albumin fraction V; all from Thermo Fisher Scientific) supplemented with LIF (Millipore) and 2i (Stemgent), Human embryonic kidney (HEK293T) cells (ATCC) were cultured in 10% fetal bovine serum (Thermo Fisher Scientific) in DMEM (Thermo Fisher Scientific).
Lentiviral Production
HEK293T cells were seeded at ˜30% confluence one day before transfection. Lentivirus were produced by cotransfecting with pHR plasmids and encoding packaging protein vectors (pMD2.G and pCMV-dR8.91) using TransIT-LT1 transfection reagents (Mirus). Viral supernatants were collected 3 days after transfection and filtered through 0.45 μm strainer. Supernatant was used for transduction immediately or kept at −80° C. for long-term storage.
High-Throughput Pooled Screening
Screens were performed in two independent replicates for both self-renewal and neural differentiation. For both screens, 108 CamES cells were transduced with the pooled lentiviral library with an MOI of 0.3, treated with puromycin, and cultured in specified medium. After a period of time indicated for each screen, cells were harvested and FACS/MACS sorted. Deep sequencing was performed to profile the sgRNA counts in each sample, and computationally analyzed to infer top sgRNA and gene hits.
Plasmid Design and Construction
To clone sgRNA vectors, the optimized sgRNA expression vector (pSLQ1373) was linearized and gel purified (Chen et al., 2013 Cell 155, 1479-1491). New sgRNA sequences were PCR amplified from pSLQ1373 using different forward primers and a common reverse primer, gel purified and ligated to the linearized pSLQ1373 vector using In-Fusion cloning (Clontech). Primers used to construct individual sgRNAs are shown in Table 1. To change the promoter of scFv-sfGFP-VP64, the EF1α and PGK promoters were PCR amplified, gel purified, and ligated to linearized pSLQ1504 using In-Fusion cloning (Clontech).
sgRNA Library Design
Putative transcription factor (TF) genes were selected according to the TRANSFAC database, and TSS (transcription start site) for each gene was determined using the Gencode and refFlat databases. All possible transcripts were selected if multiple TSSs existed for a gene. All sgRNAs targeting was −3 kb to 0 relative to TSS. Using the CRISPR-era algorithm (Liu et al., 2015 Bioinformatics 31, 3676-3678), the targeting sequences of sgRNAs adjacent to an NGG PAM (protospacer adjacent motif) were computed, starting with a G (for more efficient U6 promoter activity) with a length of 20 bp. The sgRNAs containing homopolymers spanning greater than 3 nucleotides (nt) were discarded. To avoid off-target effects, sgRNA sequences alignment to the mouse genome was computed using the short read aligner Bowtie, and those with less than 2 mismatches with another genomic region were excluded. Furthermore, sgRNA sequences that contained certain restriction sites (BstXI and XhoI) were also removed. sgRNAs with a GC content between 30% and 70% were used. An average of about 60 sgRNAs were selected for each target gene. Sequences for non-targeting negative control sgRNAs were generated using a randomized mouse gene TSS region and selected using the same rules as described above.
sgRNA Library Construction
The oligonucleotide pool was synthesized by Custom Array. The oligo library was PCR amplified, gel purified and ligated to the linearized pSLQ1373 digested with BstXI and BlpI using in-Fusion cloning.
Construction of the CamES Cell Line
Mouse ES cells were co-transduced with multiple lentiviral constructs that expressed dCas9-SunTag from a TRE3G promoter, scFV-sfGFP-VP64 from the EF1a or PGK promoter, and rtTA from the EF1a promoter. After adding Doxycycline, polyclonal cells were sorted by flow cytometry using a BD FACS Aria2 for GFP+ and mCherry+ cells. After verification of gene activation using a sgBrn2, monoclonal cells were further sorted, and one efficient cell line was selected as CamES cells.
Construction of the Tuj-1-hCD8 CamES Cell Line
Construction of CRISPR/Cas9 vector for Tuj1 knockin. The pX330-derived pSLQ1654 encoding the nuclease Cas9 and an optimized sgRNA sequence was first linearized by a BbsI digest and gel purified. Two primers sgTuj-1 F and sgTuj-1 R were phosphorylated, annealed, and ligated to the linearized vector pSLQ1654 to generate pSLQ1654-sgTuj1. sgTuj-1 F: caccgcccaagtgaagttgctcgcagc (SEQ ID NO:378). sgTuj-1 R: aaacgctgcgagcaacttcacttgggc (SEQ ID NO:379).
Construction of DNA template. The Tuj1-IRES-hCD8 vector (pSLQ1760) was assembled with three fragments (5′ homologous arm of Tuj1, IRES-hCD8 and 3′ homologous arm of Tuj1) and a modified pUC19 backbone vector by using Gibson Assembly Master Mix (New England Biolabs). Both 5′ and 3′ homology arms were PCR amplified from the genomic DNA extracted from mouse ES cells with Herculase 11 Fusion DNA polymerase (Agilent). The IRES-hCD8 was PCR amplified from pSLQ1729 (gift from Wendell Lim). The backbone vector was linearized by digestion with PmeI and Zra1. All DNA fragments and the backbone vector were gel purified followed by a Gibson assembly reaction. Primers: 5′ homologous arm F: aaagtgccacctgacactcagtccLagatgtcgtgcgg. 5′ (SEQ ID NO:380) homologous arm R: tcacttgggcccctgggct (SEQ ID NO:381). IRES-human CD8 F: caggggcccaagtgaactagtaaaattcgcccctctccctc (SEQ ID NO:382). IRES-human CD8 R: cagctgcgagcaactttaacctgcaaaaagggagcagtuaaagg (SEQ ID NO:383). 3′ homologous arm F: agttgctcgcagctggggt (SEQ ID NO:384). 3′ homologous arm R: agctggagaccgttttttctgactgactggatacagggcat (SEQ ID NO:385).
Electroporation and clonal Tuj1-hCD8 CamES cells: 2.5 μg pSLQ1654-sgTuj1, 12.5 μg Tuj1-1RES-hCD8 template DNA in 100 μL. Nucleofector solution (Amaxa) were electroporated into 1×106 CamES cells using program A-030. Both plasmids were maxiprepped using the Endofree Maxiprep Kit (Qiagen). After 3 days of culture, sorted single cells were seeded in a 96-well plate with one cell per well. All clonal cell lines were analyzed using PCR and sequencing (Yu et al., 2015 Cell 16, 142-147).
Quantitative RT-PCR
Cells were harvested using Accutase (STEMCELL), and total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN), according to manufacturer's instructions. Reverse transcription was performed using iScript cDNA Synthesis kit (Bio-Rad). Quantitative PCR reactions were prepared with iTaq Universal SYBR Green Supermix (Bio-Rad). Reactions were run on a LightCycler thermal cycler (Bio-Rad). Primers used are summarized in Table 2.
High-Throughput Pooled Self-Renewal Screening
Screens were performed in two independent replicates. For both screens. 108 CamES cells were transduced with the pooled lentiviral library with an MOI of 0.3 on day −3. On day −2, CamES cells were treated with puromycin (Invitrogen, 1 μg/mL) in basal medium supplemented with LIF and 2i. After 48 hours of puromycin selection, cells were harvested as the day 0 sample. Another 108 CamES cells with the same treatment were passaged for 10 times under the basal medium supplemented with LIF and Doxycycline (Invitrogen, 100 ng/mL), without 2i. Cells were passaged every 3 days. After 30 days, cells were harvested, stained with mouse anti-SSEA1 (BD, 1:50), and FACS sorted using BD FACS Aria2 as SSEA1+ sample (FIG. 9A). For the individual sgRNA validation experiments, a similar protocol was used, except that the CamES cells were infected with a high MOL. Top 100 hits are summarized in Table 3.
High-Throughput Pooled Neural Differentiation Screening
The neural differentiation screens were performed as two independent replicates. For both screens, 108 CamES cells were seeded at 40,000 cells/cm2 density at day −1. Cells were transduced with pooled lentiviral sgRNA library with an MOI of 0.3 at day 0 in basal medium supplemented with LIF and 2i. At day 1, puromycin was added at 1 μg/mL in ES2N medium (Millipore) with Doxycycline for another 24 hours. Fresh ES2N medium was changed with Doxycycline every day starting day 2. On day 12, cells were harvested and sorted for hCD8+ and hCD8− cells using EasySep human CD8 isolation kit (STEMCELL Technologies) (FIG. 20F). For the individual sgRNA validation experiments, a similar protocol except that CamES cells were cultured in basal medium seeded at 5,500 cells/cm2 after puromycin selection and transduced with a high MOI was used. Top 100 hits are summarized in Table 4.
Flow Cytometry Analysis
Cells were harvested, washed, and adjusted to a concentration of 106 cells/mL, in ice cold PBS with 2% FBS. Cells were stained and incubated with diluted primary antibodies at 4° C. for 30 mins in Eppendorf tubes. After staining, cells were washed three times by centrifugation at 400 g for 5 mins and resuspended in 500 μL to 1 mL in ice cold PBS. Cells were kept in dark on ice and analyzed using BD Accuri C6 Cytometer.
Immunocytochemistry
Experiments were performed on cells seeded on plate (IBIDI) that had been coated with gelatin (0.1%) overnight at 37° C. Cells were washed twice with PBS, fixed in 4% Paraformaldehyde (Wako) for 15 mins at room temperature, permeabilized and blocked with 0.1% Triton X-100, 5% donkey serum in PBS (blocking buffer) for 1 h at room temperature. After three times wash with PBS, cells were incubated with primary antibodies. The following primary antibodies with indicated dilution in blocking buffer were used: Rabbit anti-Oct4 (Santa Cruz, 1:200), Rabbit anti-Nanog (Abcam, 1:500), Mouse anti-Tuj1 (Covance, 1:1000), Rabbit anti-Map2 (Cell Signaling Technology, 1:200), Rabbit anti-NeuN (Abcam, 1:1000), Rabbit anti-vGluT1 (Synaptic Systems, 1:200). Rabbit anti-GFAP (Dako, 1:500), Rabbit anti-Olig-2 (Millipore, 1:500) Cells were incubated with primary antibodies at 4° C. for overnight, then washed three times with PBS. After staining with corresponding secondary antibodies in blocking buffer for 1 hour at room temperature, cells were washed three times with PBS and stained with DAPI (Vector Labs) for 5 mins. Washed cells were examined using a Nikon Spinning Disk Confocal microscope with TIRF.
Electrophysiology
External bath solution for whole cell patch clamp recordings contains (in mM) 140 NaCl, 5 KCl, 2 cacl2, 2 MgC2, 20 HEPES, and glucose 10, pH 7.4. Action potentials were recorded current-clamp while sodium and potassium currents were recorded under voltage clamp. The internal pipette solution contained (in mM): 123 K-gluconate, 10 KCl, 1 MgCl2, HEPES, 1 EGTA, 0.1 CaCl2, 1 MgATP, 0.3 Na4GTP and glucose 4, pH 7.2. For current clamp experiments, currents were injected to keep membrane potentials around −65 mV, and action potentials were elicited by stepwise current injections.
Western Blot
Samples were collected with NP40 buffer with protease inhibitor and phosphatase inhibitor, and boiled in 1×SDS loading buffer, separated by SDS-PAGE gels, and transferred onto a nitrocellulose (NC) membrane, which was blocked with 5% non-fat dry milk and incubated with primary antibodies at 4° C. overnight. Rabbit anti-Jun antibody (Cell Signaling Technology, 1:1000), rabbit anti-β-actin antibody (Cell Signaling Technology, 1:5000), rabbit anti-phospho-Jun antibody (Cell Signaling Technology. 1:1000) were used as primary antibodies. HRP-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 1:5000) were used as secondary antibodies. Signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). β-actin was used as a loading control.
Differentiation of Mouse ES Cells Through Embryoid Body Formation
The sgKlf2- and sgMlxip-transduced CamES cells were trypsinized, plated on ultralow attachment plates, and cultured in Knockout DMEM supplemented with 10% FBS, without Doxcycline. After 6 days, aggregated cells were collected and seeded onto gelatin-coated plates. Four days later, cells were fixed and stained with markers for three germ layers.
RNA-Seq
CamES cells were transduced with individual sgRNAs, expanded, and differentiated after 2 days of puromycin selection in 6 well plates. Total RNA was purified using RNeasy Plus Mini Kit (Qiagen). Libraries were prepared using TruSeq Stranded mRNA LT Sample Prep kit (Illumina) according to the manufacturer's instructions. Samples were combined and purified using Ampure XP Agencourt beads (Beckman Coulter) and sequenced on a Hi-Seq 4000 (Illumina), to generate paired-end 150 bp reads. Each sample was sequenced to an average depth of 40 million reads.
Reads were mapped with kallisto (Bray et al., 2016 Nature biotechnology 34, 525-527) to the provided GRCm38 downloaded from bio math at Berkley. Normalized gene expression and differentially expressed genes were estimated using sleuth (Pimentel et al., 2016 bioRxiv) and DESeq2 (Love et al., 2014 Genome Biol 15, 550) for the self-renewal and neural data, respectively. Gene ontology analysis was performed using the Bioconductor package gage (Luo et al., 2009 BMC Bioinformatics 10, 161). AP-1 targets were defined as genes that have an AP-1 consensus binding motif (Biddie et al., 2011 Mol Cell 43, 145-155; Rauscher et al., 1988 Genes & Development 2, 1687-1699; Shaulian and Karin, 2002 Nat Cell Biol 4, E131-E136; Zhou et al., 2005 DNA Research 12, 139-150) within 500 bases upstream of the TSS.
Bioinformatic Analysis of sgRNA and Gene Hits
Data processing was conducted with custom scripts. Reads were mapped allowing for a mismatch for the first and last base pair of the spacer, which uniquely identified sgRNA.
Each sample was normalized by the total read count. This gave a frequency for each sgRNA:
For the self-renewal screen, in each condition (CamES cells and SSEA+ cells), frequency for each sgRNA was averaged across replicates. sgRNA with less than 20 counts at time 0 were discarded. The sgRNA enrichment (Esg) was calculated as the log 2 fold change from the average time 0 frequency to the average SSEA+ frequency.
For the neuronal differentiation screen, the paired Tuj1-hCD8+ and Tuj1-hCD8− were used to compute the enrichment scores. Specifically, frequencies were computed as above, sgRNA with less than 1 count in the Tuj1-hCD8− library was discarded. Enrichment for each sgRNA in each replicate was calculated as the log 2 fold-change from the Tuj1-hCD8− sample to the Tuj1-hCD8− libraries. Enrichment was averaged across replicates and used as Esg in subsequent analysis.
For each gene, an enrichment score (ESgene) was calculated from the sgRNA enrichment above, as follows. An unnormalized enrichment score (Egene.top3) was calculated by averaging Fsg for the 3 sgRNA with highest Esg. Egene.top3 was normalized by the distribution of nontargeting sgRNA as follows (Gilbert et al., 2014 Cell 159, 647-661).
Suppose a gene had N targeting sgRNA. Then, 10000 bootstrap samples of size N were drawn from the nontargeting sgRNA. For each sample of size N, Esample.top3 was computed as above. This gave an empirical estimate of the distribution of Egene.top3 if the all the sgRNA targeting that gene had been negative control sgRNA. For the final, normalized gene enrichment score (ESgene), the unnormalized enrichment score was divided by the 0.9 quantile of thie smpirical distribution:
After ranking genes by ES, the most enriched sgRNA for each gene was selected to subsequently validate.
Results Generation of CRISPRa Mouse Embryonic Stem Cells for Single sgRNA-Mediated Gene Activation and Cell Fate Control
Single sgRNA-mediated efficient endogenous gene activation is useful for large-scale pooled screens of sophisticated cell differentiation phenotypes (FIG. 1A). To establish such a highly efficient CRISPRa system, a reported CRISPRa system based on a polypeptide array, SunTag, was used (Tanenbaum et al., (2014). Cell 159, 635-646). A panel of individual or mixed sgRNAs was used to activate endogenous Brn2 (FIGS. 8A and 8B), a gene driving neuron formation in mouse ES cells (Sokolik et al., 2015 Cell Systems 1, 117-129). Mixed sgRNAs showed better activation compared to individual sgRNAs, whereas none of them induced neural differentiation.
The dCas9-SunTag system contains two components, a SunTag polypeptide domain fused to dCas9 and a VP64 transactivator domain fused to a single chain fragment variable (scFv). It was investigated whether their expression ratio was a key factor determining the activation efficiency. To facilitate fine-tuning their ratio, each component was cloned onto a lentiviral vector (FIG. 8A). The dCas9-Suntag fusion was expressed using a Doxycycline (Dox)-inducible promoter pTRE3G, and the SFFV promoter was replaced with an EF1a promoter for Tet-On 3G transactivator expression, as silenced SFFV activity was observed during ES cell differentiation. It was tested if promoters (PGK, EF1a, and SFFV) with different strengths driving scFv-VP64 fusion could lead to various activation efficiencies. It was observed the PGK promoter exhibited best endogenous Brn2 expression using both bulk and clonal cells (FIGS. 8C and 8D). By tuning the stoichiometry ratio between the two components, an enhanced CRISPRa (eCRISPRa) system with better activation of endogenous genes was obtained.
Twenty eight clonal cell lines with the PGK promoter were sorted, and one cell line (#5) showing best Brn2 activation was obtained, which was named CamES (CRISPR-activating mouse ES) cells (FIG. 8E). It was confirmed that this cell line could be stably cultured in ES cell conditions, while maintaining stem cell morphology and pluripotency and expressing eCRISPRa components over a long-term passage (FIG. 8F). It was determined if CamES cells allowed efficient activation of another gene, Asc11, using a single sgRNA. All 5 Asc11 sgRNAs showed strong activation (>10,000 fold) compared to using a control sgRNA (FIG. 1B). In addition, the activation efficiency varied among 5 sgRNAs, showing that a broad range of gene activation can be achieved.
It was next tested if this promoted neural differentiation (Chanda et al., 2014 Stem Cell Reports 3, 282-296). Using a single sgAsc11, robust differentiation of CamES cells into a neuronal phenotype was observed at day 8, which stained positively for the neuronal markers Tuj1 (class III beta-tubulin) and Map2 (Microtubule-associated protein 2) (FIG. 1C). All negative controls (CamES cells without sgRNA, CamES cells with non-target control sgRNA, and E14 mouse ES cells with sgAsc11) showed no neural differentiation morphology or neural marker expression, confirming neurons were indeed induced by eCRISPRa-mediated target gene activation. Another neural transcription factor, Neurog1 (Velkey and O'Shea, 2013 Dev Dyn. 242, 230-253), was tested with a single sgRNA, and similarly observed neuron formation (FIG. 1C). The cell line also showed efficient skeletal muscle differentiation using a single sgRNA activating MyoD1 (FIG. 1C) (Shani et al., 1992 Symp. Soc. Exp. Biol. 46, 19-36). These experiments together demonstrate that CamES cells allow single sgRNA-mediated endogenous gene activation and cell differentiation.
The CamES cells activating endogenous Asc11 were compared with overexpression of exogenous Asc11 cDNA for neural differentiation. A similar neuronal phenotype was observed using the two approaches (FIG. 1C). It was found that cells using two systems showed similar morphogenetic features characterized by the formation of neural rosettes after 6 days of differentiation and extensive neurite outgrowth between days 8-12 (FIG. 9H). Though overexpression of exogenous cDNA showed higher total Asc11 expression, CRISPRa-mediated endogenous Asc11 activation exhibited comparable or even better neural differentiation as seen by the fold change of other neural markers Brn2, Tuj1, and Map2 over a 10-day differentiation process (FIG. 1D). The data demonstrated that modulating endogenous genes is a better strategy for directed cell differentiation compared to cDNA expression. Taken together, these results showed that the CamES cells were able to induce high-level endogenous gene expression using only a single sgRNA for controlling cell fate.
CamES Cells Allow an eCRISPRa-Mediated Dropout Screen to Identify Transcription Factors that Maintain Self-Renewal
CamES cells were used as an unbiased screening platform to identify key factors among the set of all putative transcription factors that direct cell fate determination. Initial studies focused on factor contributing to the maintenance of ES cell self-renewal. An sgRNA library targeting all putative TFs (˜800) and a small set of lincRNAs (long intergenic noncoding RNAs) (˜50) was generated. Multiple sgRNA (60 sgRNAs per gene on average) were designed to target each gene to cover a broad range of gene activation. An additional 9,296 non-targeting negative control sgRNAs were included. Altogether, a library with a total of 55,336 sgRNAs was generated (FIG. 2A).
The sgRNA library was introduced into CamES cells as a gain-of-function screen to study stem cell self-renewal. Self-renewal of mouse ES cells in serum-free conditions requires simultaneous inhibition of the GSK3 and ERK pathways, which is typically achieved by using two small molecule inhibitors (2i) (Ying et al., 2008 Nature 453, 519-523). It was determined whether activating transcription factors could functionally rescue the loss of 2i to support self-renewal over a long period of time. To do this, the lentiviral sgRNA library was transduced into CamES cells, cultured the transduced cells in −2i medium, and passaged every three days (FIGS. 2A and 9A). For library transduction, MOI (multiplicity of Infection) was kept below 0.3 such that the majority of cells were transduced only with a single sgRNA. Over half of cells quickly lost pluripotency markers (SSEA1 and Oct4) and initiated spontaneous differentiation within two passages post library transduction (FIGS. 28 and 2C). Repeated passaging of cells removed most differentiated cells, while the SSEA1+ population gradually increased over time, providing a dropout screen. After 10 passages, SSEA1+ cells were sorted using FACS (flow cytometry activated sorting), which further increased SSEA1+ cell percentage to 96.9% (FIG. 2B). The sorted cells showed mouse ES cell morphology and were Oct4+, confirming maintenance of pluripotency (FIG. 2C).
To identify genes whose gain-of-function maintains self-renewal of ES cells, deep sequencing was used to read out the sgRNA representation (FIG. 2A). The overall distribution of sgRNAs from samples collected from the original plasmid library, CamES cells with sgRNA library at day 0, and sorted SSEA1+ cells after passage 10 were compared (FIG. 9A). Only a small fraction of sgRNAs were detected after sorting compared to the plasmid library and day 0 samples (FIGS. 2D and 2E), indicating an efficient selection process.
Gene-level enrichment scores were obtained by considering the enrichment of the top three sgRNAs targeting each gene and normalizing by the empirical distribution of the non-targeting sgRNA. A good correlation was obtained between both sgRNA enrichment and gene-level scores across independent library transductions (FIG. 9B).
Validation of Top Enriched sgRNAs Promoting Long-Term Maintenance of Self-Renewal in ES Cells
Using the non-targeting sgRNA normalized gene scoring method, all detected sgRNAs and their targeting genes were ranked (FIG. 10). For each gene, the majority of designed sgRNAs were depleted, implying either most genes had no function in self-renewal or the depleted sgRNAs were unable to sufficiently activate gene expression for functional genes. Major pluripotency factors such as Nanog, Sox2, Klf4, and Oct4 appeared as top enriched hits, consistent with previous works showing their critical roles in maintaining stem cell self-renewal (Chambers et al., 2003 Cell 113, 643-655; Masui et al., 2007 Nat. Cell Biol. 9, 625-635; Mitsui et al., 2003 Cell 113, 631-642; Niwa et al., 2000 Nat. Genet. 24, 372-376; Zhang et al., 2010 J. Biol. Chem. 285, 9180-9189).
The most enriched sgRNAs of the top 18 genes were selected for validation (FIG. 3A). The 18-gene list contained pluripotency genes (Klf2 and Id1) (Jiang et al., 2008 Nat. Cell Biol. 10, 353-360; Yeo et al., 2014 Cell Stem Cell 14, 864-872; Ying et al., 2003a Cell 115, 281-292), lineage specific genes (Etv2 and Isl2) (Koyano-Nakagawa et al., 2012 Stem Cells 30, 1611-1623; Thaler et al., 2004 Neuron 41, 337-350), and one lincRNA gene (4930555M17Rik). For validation, 18 individual sgRNAs were constructed and transduced into CamES cells. Six individual non-targeting sgRNAs were included as negative controls. None of the negative control sgRNAs was able to maintain stem cell self-renewal in −2i medium condition beyond passage 2.
Quantitative PCR results confirmed activation of target genes by each sgRNA (FIG. 3B). All 18 sgRNAs maintained stem cell morphology and expressed pluripotency markers Oct4, Nanog, and SSEA1 after culturing in −2i condition over 30 days (FIG. 3C). Notably, 9 out of 18 validated genes (Mlxip, Etv2, Zc3h11a, Zfp36, Isl2, Tfeb, Fig1a, Hsf2, and Hoxc11) are not previously annotated for maintenance of pluripotency and self-renewal. The high rate of validated true hits indicates that the screening method provides an effective dropout screen of genes promoting self-renewal and maintaining pluripotency.
Deep Sequencing and Functional Validation Confirmed the Function of Positive Hits for Self-Renewal Maintenance
sgMlxip was chosen to explore its role in promoting self-renewal. The MLXIP protein forms a heterodimer with MLX (Max-like protein X) and modulates transcriptional regulation in response to cellular glucose levels (Stoltzman et al., 2008 Proc. Natl. Acad. Sci. USA 105, 6912-6917), and its function related to ES cell self-renewal is unknown.
The developmental potential of CamES +sgMlxip cells cultured in −2i conditions for generating the three germ layers was evaluated using CamES +sgKlf2 as a comparison. After removal of Dox to switch of eCRISPRa activity, spontaneous differentiation of both samples in serum-based medium via embryoid body formation generated cells representative of ecdoderm (Tuj1+), mesoderm (SMA+), and endoderm (Sox17+) lineages (FIG. 4A). This confirmed the differentiation potential of these cells cultured in −2i medium.
RNA-seq analysis was performed on CamES +sgMlxip and CamES +sgKlf2 cells cultured in −2i conditions, and compared to CamES cells cultured with or without 2i. Both samples exhibited high mRNA expression for most pluripotency genes and low expression for most lineage specific genes, with a pattern similar to ES cells cultured in 2i medium and distinct from cells without 2i (FIG. 11A), indicating that the CamES +sgMlxip and CamES +sgKlf2 cells maintained a similar gene expression profile as the undifferentiated stem cells in 2i medium.
The 2i cocktail contains two small molecules that maintain pluripotency by inhibiting GSK3 (CHIR99021) and MEK1/2 (PD0325901) (Ying et al., 2008 Nature 453, 519-523). Via activation of the Wnt pathway and inhibition of the MAPK pathway, the 2i molecules inhibit differentiation while promoting proliferation of ES cells. The RNA-seq gene expression profiles for the Wnt and MAPK pathways were compared among the samples. For the Wnt pathway genes, CamES-sgMlxip cells correlated well with CamES cells in +2i medium (R2=0.81), while poorly with CamES cells in −2i medium (R2=0.35) (FIG. 4B). A different ratio distribution of corresponding gene expression between +sgMlxip/+2i and +sgMlxip/−2i was found (FIG. 11B) (Zhang et al., 2013 Stem Cells 31, 2667-2679).
Similar results were observed for the MAPK pathway: there was a good correlation between CamES +sgMlxip and CamES +2i samples (R2=0.91), compared to a poor correlation between CamES-sgMlxip and CamES-2i (R2=0.59). Gene expression related to the MAPK pathway showed a similar pattern at the transcript level in both CamES +sgMlxip and CamES +2i cells. For example, inhibition of Jun, a major transcription factor of the MAPK pathway, was observed in both CamES +sgMlxip and CamES +2i cells, as well as inhibition of other MAPK related genes (EGF, FAS, FGF, PDGF and TGFb) (FIG. 11C). These results together indicate that CamES +sgMlxip cells possess similar Wnt and MAPK pathway activities as CamES +2i cells.
The PI3K pathway, which is important in the regulation of ES cell pluripotency and proliferation (Yu and Cui, 2016 Development 143, 3050-3060), was also investigated. The CamES +sgMlxip cells also showed a similar expression pattern as CamES +2i cells (FIG. 4D). For example, PI3K-related genes such as Fos, Mapkapk2, Gadd45b, and Gadd45g were downregulated in both CamES +sgMlxip and CamES +2i cells, while Ccnd1, Cdk2, Cdk9, and Sod2 were similarly upregulated (FIG. 4D). The PI3K gene expression further confirms the similarity between CamES +sgMlxip cells and ES cells cultured in 2i medium.
In summary, both functional tests and gene expression indicate that true positive hits identified using the CRISPRa screening method maintain self-renewal of stem cells.
Engineered CamES Cells Allow an eCRISPRa-Mediated Non-Dropout Screen to Identify Key Factors Promoting Neural Differentiation
A eCRISPRa gain-of-function screen was performed to identify TFs that promote the dynamic, complex neural differentiation process. Transcription factor-mediated lineage specification is heterogeneous and stochastic: unlike in the dropout screen, a desired differentiated cell type may only represent a small subset of the total population; and spontaneous differentiation may generate the desired cell type even when a non-functional factor is present.
To address these challenges, a clonal reporter CamES (Tuj1-hCD8 CamES) cell line carrying a biallelic human CD8 (hCD8) gene cassette appended downstream to endogenous Tuj1 via an IRES (internal ribosome entry site) was established (FIGS. 5A and 12A). Upon transduction with sgAsc11 and Dox induction, differentiated Tuj1-hCD8 CamES cells expressed both Tuj1 and hCD8 (Figure S5B). MACS (magnetic-activated cell sorting) was used to isolate hCD8+ and hCD8− cells, and observed hCD8+ cells expressed a higher level of neural markers (Tuj1 and Map2) compared to hCD8− cells and unsorted cells (FIG. 5B). This demonstrates that sorted hCD8+ cells are positively correlated with differentiated neuron cells.
The parameters of cell density and differentiation time for screening, which affected neural differentiation efficiency, were determined. 40,000 cells/cm2 was chosen as the seeding density, as Tuj1-hCD8 CamES cells transduced the sgRNA library maximized the seeding cell number and showed detectable neural marker expression Tuj1 and Map2 (FIG. 12C). Day 12 was chosen as the sample collection time point, when differentiated cells showed neuronal morphology and expression of neural markers (FIGS. 12D and 12E). With these conditions, MACS was performed to sort and isolate Tuj1-hCD8 positive and negative populations (FIGS. 5A and 12F).
Deep sequencing was used to identify sgRNAs for transcription factors that enhance neural differentiation. The overall distributions of sgRNA from samples collected from plasmid library, sorted Tuj1-hCD8+ and Tuj1-hCD8− cells was compared (FIGS. 5A and 12F). In contrast to the self-renewal screen, a larger fraction of sgRNAs were detected after sorting compared to the plasmid library (FIGS. 5C and 5D). In addition, Tuj1-hCD8+ and Tuj1-hCD8− cells exhibited similar sgRNA depletion.
Stem cell differentiation is affected by stochastic factors. In these experiments, activation of Asc11, a powerful neural inducer, led to only 47.6% of cells being Tuj1-hCD8+(FIG. 12B). In addition, the effects of spontaneous differentiation and less proliferative capacity of desired differentiated cells may affect the overall screening outcome. Thus, most sgRNAs in the non-dropout neural differentiation screen cannot be depleted as strongly as in the dropout screen (FIGS. 5C and 5D). It was contemplated that normalizing positive population against the negative population would more accurately identify the TFs that drive neural differentiation. Thus, paired comparative analysis of Tuj1-hCD8+ and Tuj1-hCD8-cell populations was used to rank the most enriched genes and their sgRNAs.
Validation of Top Enriched sgRNAs Promoting Neural Differentiation
Among the ranked gene hits, the top 20 most effective sgRNAs were chosen for validation (FIGS. 13 and 6A). The 20-gene list contained known neuron-driving transcription factors (Neurog1, Brn2, and Klf12) (Theodorou et al., 2009 Genes Dev. 23, 575-588), and genes that were not previously linked to neural early development including epigenetic regulators (Ezh2, Suz12) and signaling proteins (Jun).
Twenty individual sgRNAs for the top gene hits, as well as 6 non-targeting negative control sgRNAs were tested, Quantitative PCR results showed activation (10 to 10,000 fold) of 19 genes out of 20 tested by their cognate sgRNA (FIG. 6B). Using Tuj1-hCDg CamES cells, Tuj1-hCD8 expression was measured after 12 days of differentiation in basal medium by FACS. All 20 sgRNAs transduced-cells showed expression of hCD8 in a significant percentage of cells (10-50%), while all 6 negative control sgRNAs or cells without a transduced sgRNA showed no hCD8+ cells (FIG. 6C).
Another neuronal marker, NCAM, was used to test differentiation of CamES cells. Similarly, all 20 sgRNAs generated NCAM+ cells (20-60%) after 12 days of differentiation in basal medium, and all negative control sgRNAs showed much less NCAM+ cells (below 10%) (FIG. 6D). Positive immunostaining of neural marker Map2 in all 20 sgRNAs differentiated cells was observed (FIG. 6E). One sgRNA targeting Arnt failed to activate target expression at the time it was assayed for activation. However, this sgRNA was able to induce neural differentiation, which may be due to a longer latency of activation, activation of nearby regulatory elements (e.g., a cis-acting lincRNA), or off-target effects.
Activation of different endogenous genes induced different neural subtypes (FIG. 6F). Most genes induced a high percentage cells expressing neuron markers (Tuj14+, Map2+, and NeuN+). Some hits such as Nr2f1, Nr3c1, and Tcf15 induced more cells with a positive astrocyte marker GFAP. The oligodendrocyte marker Olig2 and the Glutamatergic neuron marker vGluT1 were assayed, and varying levels of expression across the top 20 sgRNAs was observed.
Functional Test and Transcriptome Profiling Confirmed sgJun-Induced Neural Differentiation
The role of Jun for promoting neural differentiation was examined. Jun has not previously been tied to early neural development. It was observed that sgJun could induce functional neurons that were able to generate action potentials upon current injection (FIG. 7A). RNA-seq was performed to profile the transcriptome of CamES +sgJun cells at various time points (day 0, 2, 5, and 12) (FIG. 14A). Cells were analyzed at different time points using PCA (Principal component analysis), and four distinct clusters that correlated with a dynamic process of neural differentiation were identified (FIG. 7B). It was found that the pluripotency genes were consistently downregulated starting at day 2 after sgJun transduction, and neural marker genes were upregulated throughout the process (FIG. 7C). Meanwhile, day 12 cells were highly enriched for Gene Ontology (GO) terms associated with neural fate and functions, such as axonogenesis and neuron projection guidance (FIG. 7D).
Jun regulates downstream target genes through its phosphorylation and the AP-1 complex formation with c-Fos (Rauscher et al., 1988 Genes Dev. 2, 1687-1699). It was confirmed that endogenous Jun induced by sgJun also was phosphorylated (FIG. 7E). Analysis of AP-1 target genes showed that they were activated at days 5 and 12 (FIG. 7F). It was also found that expression of both FGF ligands and receptors (Fgf5, Fgf8, Egf9, Fgfr1, Fgfr2, and Fgfr3) were rapidly increased at day 2 (FIG. 14B). Meanwhile, key genes of the Wnt pathway (Wnt3a, Wnt6, Wnt10b, and β-catenin) were also upregulated in sgJun-induced cells at days 5 and 12 (FIGS. 7G and 14B).
Previous work reported that overexpression of β-catenin in mouse ES cells induce neurogenesis (Otero et al., 2004: Development 131, 3545-3557). The excessive expression of Wnt genes in the cells indicates that the Wnt pathway plays an important role in sgJun-induced neurogenesis (FIG. 14C). Furthermore, since MAPK, the downstream pathway of FGF, activates Jun via phosphorylation, sgJun-activated endogenous Jun likely maintains its stable expression and sustained activity via a FGF/MAPK positive feedback loop (FIG. 14C), which is consistent with works showing the important role of FGF/MAPK pathway in neural fate commitment of ES cells (Chen et al., 2010 Journal of Biomedical Science 17, 1-11; Ying et al., 2003b Nat. Biotechnol. 21, 183-186). Together, modulation of these pathways through endogenous Jun activation indicates a functional role of Jun for induced neural differentiation of mouse ES cells.
Paired-Analysis is Useful in the Non-Dropout Cell Differentiation Screen
In dropout screens, cells that are negative for the phenotype of interest are almost completely removed from the selected population. Therefore, one can calculate enrichment of the selected population relative to initial pool of sgRNAs to infer functional genes (FIG. 14D). In non-dropout screens, the phenotype of interest may arise stochastically (FIG. 14D). If activation of a gene confers a proliferative advantage, then even if the probability of the phenotype of interest is small (spontaneous differentiation), with more cells it would appear that the gene is enriched in the selected population when compared to the initial population. In fact, a high correlation of enriched genes between the positive and negative Tuj1-hCD8 populations was found (FIG. 14E). The top hits relative to initial sgRNA pool in both populations contain many proliferative genes, but few are related to neural phenotype (FIG. 14F). Those proliferative genes disappear, and several known neural genes are identified when the Tuj1-hCD8+ population was normalized against Tuj1-hCD8− population. The final rankings show little correlation with the enrichment in the positive population (FIGS. 14E and 14F), indicating that these proliferative genes were mostly false positives.
TABLE 1
Primers used to construct individual sgRNAs.
Primers sgRNA sequence SEQ ID NO
Forward gtatcccttggagaaccaccttgttgnnnn 386
primer nnnnnnnnnnnnnnnngtttaagagctaag
ctggaaacagca
Reverse gatcctagtactcgagaaaaaaagcaccga 387
primer ctcggtgccac
sgBrn2-1 gggagagagcttgagagcgc 388
sgBrn2-2 gcccaggcgcgtgccgctgcgag 389
sgBrn2-3 gcggtatccacgtaaatcaaa 390
sgBrn2-4 gctccggtctgggaggttgctag 391
sgBrn2-5 gcaccaatcactggctccggtc 392
sgBrn2-6 gactgagaagactgggcgcccg 393
sgBrn2-7 gaatctgaatcgctgagcta 394
sgBrn2-8 gaggccggggacagaagaga 395
sgBrn2-9 gagcgcctggaccgaccgcc 396
sgBrn2-10 gaaatcgtagtcctgctggctgact 397
sgBrn2-11 gtgtgtgtgttcctaggagaa 398
sgBrn2-12 gtctagctttggctctcgttct 399
sgAscl1-1 ggctgggtgtcccattgaaa 400
sgAscl1-2 gaatggagagtttgcaaggag 401
sgAscl1-3 gtctggagggaaaagtgtctt 402
sgAscl1-4 gagttactgcggagagaagaaa 403
sgAscl1-5 gagggaaaggctgctcagaca 404
Neurog1 ggctgctgggagttgtgcaa 405
Myod1 ggtctccagagtggagtccg 406
Nanog ggaagtttcaggtcaagtgg 407
Mlxip ggcactccacgtggtgggta 408
Sox2 gcctttgcaccctttggatg 409
Klf2 gagggtaatagagagaggga 410
Etv2 gttcgtggctcacctctggc 411
Klf4 gtgcgtatgcgagagagggc 412
Zc3h11a gcattatcccttagatgcca 413
Hsf2 ggattcgcatggaaagggtt 414
Hey2 ggtgtgtctagacaggagac 415
ZFP36 ggttgtgtacgaccaactgg 416
Isl2 gagaggagaaaggagagggt 417
Tfeb gacatgggcaataacagggt 418
Nobox gcctgcttgatggaaaggta 419
Figla ggcatctgaaaccaggagga 420
Bcl6 ggtgggaagagagagagaga 421
Id1 ggctcaagaactgaaagggt 422
Hoxc11 ggaggagagagagagagggt 423
M17Rik gctgataaggtagaaaggta 424
Foxo1 ggttcaggatgagtggaggc 425
Nr2f1 ggagccaagagaagggctgc 426
Rb1 ggctacatacagtctaggtt 427
Pou3f2 gaggaaggactgagaagact 428
Ezh2 ggttcctttcggcaccttgg 429
Maz ggaaggcatctctgggaagc 430
Nr4a1 gctaacgtgtagtctcgttg 431
Arnt gtttgaaactccaggttaat 432
Dmrt3 gaggagttgatagttgttcc 433
Sin3b gtgcaagaattcagtccaca 434
Jun gagaataaagtgttgtgccg 435
Suz12 gaagctctcaaggcgagaaa 436
Klf12 gatttgaccatctcttgccg 437
Nr3c1 gtcactgctctttaccaaga 438
Tcf15 gggatatgctcactttggga 439
Zeb1 gaaggaactaagtttcttct 440
Nr6a1 gatgacggtcggccgtagtt 441
Mecom gattctcaggcagggctcta 442
Hoxc8 gctctttcctctaacagccc 443
TABLE 2
Primers used for quantitative PCR.
SEQ
Gene name Primer sequence ID NO
RiboL7 F accgcactgagattcggatg 444
RiboL7 R gaaccttacgaacctttgggc 445
Ascl1 F aagaagatgagcaaggtggagacg 446
Ascl1 R gagatggtgggcgacagga 447
Brn2 F tttcctcaaatgccctaagc 448
Brn2 R ggaggggtcatccttttctc 449
Tuj1 F agtcagcatgagggagatcg 450
Tuj1 R agtcccctacatagttgccg 451
Map2 F agcactgattgggaagcact 452
Map2 R caattcaaggaagttgtaaagtagtgaag 453
tttg
Nanog F aaccaaaggatgaagtgcaagcgg 454
Nanog R tccaagttgggttggtccaagtct 455
Mlxip F aagctcttcgagtgcatgac 456
Mlxip R ttgttgagccggatcttgtc 457
Sox2 F acaagagaattgggaggggt 458
Sox2 F ttttctagtcggcatcaccg 459
Klf2 F ccttcggtcttttcgagga 460
Klf2 R cttggcctccagcagctc 461
Etv2 F acgtagaaggctgctggaa 462
Etv2 R tgtccagtctcgcgacca 463
Klf4 F aaaagaacagccacccacac 464
Klf4 R cgtcccagtcacagtggtaa 465
Zc3h11a F catcggttcggtaaagtttctgt 466
Zc3h11a R ccactcagccacagaaatcg 467
Hsf2 F tgaagcagagttccaacgtg 468
Hsf2 R ttgctcatccaagaccagaa 469
Hey2 F tgaagatgctccaggctaca 470
Hey2 F tctgtcaagcactctcggaa 471
Zfp36 F tctcttcaccaaggccattc 472
Zfp36 R tatgttccaaagtcctccga 473
Isl2 F agtcgaggtgcagacgtac 474
Isl2 R ttgcctagggagcctgact 475
Tfeb F caacagtgctcccaacagtc 476
Tfeb R ttgatgtagcccagcacgc 477
Nobox F acggagaagctctgcaagaa 478
Nobox R ttgtcttgatcatcctggatgg 479
Figla F actcggctgtgttctggaag 480
Figla R tgggtagcatttcccaagag 481
Bcl6 F ttggactgtgaagcaaggca 482
Bcl6 R actccggaggcgattaagg 483
Id1 F ctgaacggcgagatcagtg 484
Id1 R tttcctcttgcctcctgaag 485
Hoxc11 F aacacgaatcccagctcgt 486
Hoxc11 R ggatctggaatttcgaataagggc 487
M17Rik F cctgagactaatactgtatgatttggaaa 488
M17Rik R cacaggtttagagataaccaaagtgg 489
Foxo1 F gagtggatggtgaagagcgt 490
Foxo1 R tgctgtgaagggacagattg 491
Nr2f1 F ccaacaggaactgtcccatc 492
Nr2f1 R attcttcctcgctgaaccg 493
Neurog1 F cggcttcagaagacttcacc 494
Neurog1 R ggcctagtggtatgggatga 495
Rb1 F gcagcatcttgattctggaac 496
Rb1 R tgtcaagttggcttccacttt 497
Pou3f2 F tttcctcaaatgccctaagc 498
Pou3f2 R ggaggggtcatccttttctc 499
Ezh2 F acttctgtgagctcattgcg 500
Ezh2 R cgactgcattcagggtcttt 501
Maz F gtggcaagatgctgagctc 502
Maz R cattggacaaacctcaccagtac 503
Nr4a1 F gctagaaggactgcggagc 504
Nr4a1 R attgagcttgaatacagggca 505
Arnt F ggcgactacagctaacccag 506
Arnt R gccctctgtacaacagctcc 507
Dmrt3 F agcgcagcttgctaaacc 508
Dmrt3 R gcttttgacaacatctgggg 509
Sin3b F agagttcggacagttcctgc 510
Sin3b R tcctcattcttctgcccact 511
Jun F gaaaagtagcccccaacctc 512
Jun R aatcagacaggggacacagc 513
Suz12 F tcgaaattccagaacaagca 514
Suz12 R tgtggaagaaaccggtaaatg 515
Klf12 F ccataaagaatctcagcgcc 516
Klf12 R ccatatcggggtagttgtgg 517
Nr3c1 F ggacaacctgacttccttgg 518
Nr3c1 R ctggacggaggagaactcac 519
Tcf15 F tctgcaccttctgtctcagc 520
Tcf15 R aaccagggatccaggttcat 521
Zeb1 F acagagaatggaatgtatgcatgtg 522
Zeb1 R agattccacactcgtgaggc 523
Nr6a1 F gcaacggtttctgtcaggat 524
Nr6a1 R ggttcgttgttcagctcgat 525
Mecom F acagcatgagatccaaaggc 526
Mecom R ttatcccatctgcatcagca 527
Hoxc8 F aaatcctccgccaacactaa 528
Hoxc8 R tgtaagtttgtcgaccgctg 529
TABLE 3
Top 100 gene hits from CRISPRa self-renewal screen.
Rank Gene name Enrichment score
1 Nanog 6.436538099
2 Sox2 5.110480488
3 Klf4 4.679609611
4 Bc16 4.250485879
5 Tfeb 4.160094948
6 Mlxip 3.992616854
7 Klf2 3.911099626
8 Etv2 3.644806172
9 Isl2 3.468873873
10 Hey2 3.189713541
11 Zfp36 2.929649816
12 Zc3h11a 2.83067826
13 Sox18 2.813521887
14 Nobox 2.627399442
15 Figla 2.607875769
16 4921504A21Rik 2.594074135
17 Hsf2 2.552027639
18 Hoxc11 2.518020583
19 Tfcp211 2.460616651
20 Spi1 2.383834061
21 Id1 2.277631872
22 Tlx2 2.237444329
23 4930555M17Rik 1.890123174
24 Nov 1.874188087
25 Klf5 1.852149928
26 Crygf 1.829064836
27 Sox11 1.807058979
28 Atf5 1.774675631
29 Esrrg 1.746829472
30 Tsn 1.744364085
31 Thrb 1.602037631
32 Nfe212 1.593264465
33 Lhx1 1.548863185
34 Pou5fl 1.518892786
35 Ebfl 1.452433199
36 Dlx5 1.403820123
37 Mycl 1.371065103
38 Atfl 1.36137151
39 Tftdp1 1.326446848
40 Irx6 1.194061551
41 Zfp2 1.191847857
42 Nfatc1 1.188011066
43 Crem 1.049272101
44 Nr3c1 1.042323412
45 Pax5 1.024334324
46 Foxfl 1.00419091
47 Snai1 0.960150521
48 Zfp423 0.947760908
49 Esrrb 0.904004441
50 Pbx2 0.899430618
51 Foxd4 0.895608808
52 Sox1 0.878521398
53 Lbx1 0.841046411
54 Mecom 0.820757135
55 Ncor2 0.780231219
56 Nr0b2 0.752404477
57 Trp53 0.743632306
58 Lmo3 0.732198452
59 En1 0.731989985
60 Rfx1 0.725576385
61 Maz 0.700348134
62 Alx4 0.686172162
63 Nr1d2 0.679722158
64 Tcf15 0.620553011
65 Egr3 0.617223131
66 Nr5a1 0.614144289
67 Tfe3 0.60710143
68 Spdef 0.593267507
69 Tcfl2 0.564881228
70 Dlx2 0.541542994
71 Vezfl 0.534712227
72 Gata1 0.504194994
73 Arf6 0.491842327
74 Sox21 0.477561748
75 Lmx1a 0.447377739
76 Pou4f2 0.410773196
77 Nr1b2 0.406668822
78 Fox11 0.394295617
79 Stat5b 0.369982068
80 Evx2 0.360115239
81 Sox5 0.348850095
82 Hivep3 0.324844194
83 Tfap2a 0.303852954
84 Glis3 0.277004435
85 Mafk 0.265635614
86 Hoxb5 0.256534563
87 Myf5 0.252944449
88 Nkx2-5 0.251102596
89 Lhx6 0.244502182
90 Foxs1 0.242003106
91 Rnps1 0.2417908
92 Mitf 0.229103445
93 Drd1a 0.21477535
94 Lmx1b 0.191984237
95 Vax2 0.183188363
96 Hoxa11 0.1661187439
97 Otp 0.163494265
98 Mxd4 0.160929842
99 Plag11 0.137545433
100 Smad5 0.128584689
TABLE 4
Top 100 gene hits from CRISPRa neural differentiation screen.
Rank Gene name Enrichment score
1 Foxo1 2.49122811
2 Nr2fl 2.448600182
3 Neurog1 2.43849068
4 Rb1 2.435300527
5 Pou3f2 2.385360453
6 Ezh2 2.380072461
7 Maz 2.361103604
8 Nr4a1 2.351837703
9 Arnt 2.317336958
10 Dmrt3 2.304207908
11 Sin3b 2.280599668
12 Jun 2.277732884
13 Suz12 2.276236754
14 KIfl2 2.269476929
15 Nr3cl 2.249983644
16 Tcfl5 2.229200027
17 Zeb1 2.221200461
18 Nr6a1 2.208496165
19 Mecom 2.207944981
20 Trim24 2.206262504
21 Hoxc8 2.184103377
22 Foxk1 2.171388615
23 2410080102RiK 2.171161939
24 Nr4a3 2.168779599
25 Trp73 2.16579857
26 Foxs1 2.162897697
27 Ikzf3 2.15938851
28 Nkx2-6 2.15063949
29 Sox11 2.140964961
30 1110054M08Rik 2.139005342
31 Crem 2.133968618
32 Meis3 2.131453549
33 Bmyc 2.130409666
34 Epas1 2.129339686
35 Nr2f6 2.128397081
36 Nacc1 2.120269011
37 Bsx 2.120136772
38 Foxd3 2.114601186
39 Myog 2.107435864
40 Smad3 2.105254748
41 Wt1 2.091731056
42 Taz 2.091306567
43 Smad7 2.071136269
44 Stra13 2.06971649
45 Hoxc4 2.062634453
46 Pou3f3 2.058607569
47 Zbtb12 2.051837502
48 Atf5 2.042025795
49 Gtf2a2 2.041587014
50 Pura 2.040735147
51 Snai1 2.040229657
52 Ncor1 2.038396405
53 Pcbp2 2.036271048
54 E2f2 2.028758908
55 Nfkbib 2.023153101
56 Gli2 2.021010016
57 Nr0b1 2.020715359
58 B230110C06Rik 2.016733057
59 T 2.014396786
60 Runx3 2.011724145
61 Rxra 2.011600497
62 Mafk 2.009964981
63 Foxnl 2.006315586
64 Smad4 1.999197443
65 Meis2 1.998728368
66 Hoxa1 1.996287157
67 Zic1 1.992579239
68 Sebox 1.99248237
69 Nfyc 1.983084664
70 Lmx1b 1.980716237
71 Lhx3 1.979175342
72 Hmx2 1.978886945
73 Arf6 1.977331424
74 Nfatc3 1.975872129
75 Neurod6 1.973516686
76 Smarca4 1.972359038
77 Twist1 1.971479015
78 Gzfl 1.963483117
79 Hoxcl0 1.962998475
80 Tbx4 1.962626034
81 Npas2 1.962608209
82 Ctbp1 1.960624385
83 Gcm2 1.960206991
84 Is12 1.957324105
85 Arid5a 1.956887379
86 Lef1 1.955552772
87 RP24-399L6.2 1.953337042
88 Smad5 1.949029539
89 Lbx1 1.948838891
90 Pax3 1.945680745
91 Foxj1 1.944149198
92 Tbx5 1.943975816
93 Barh11 1.943598679
94 Hoxd11 1.9410811
95 Pou1fl 1.939557398
96 Klf3 1.938997548
97 Pcbp1 1.937292841
98 Evx2 1.935442174
99 Irx5 1.934100096
100 Nkx6-3 1.928635054
Example 2 Quantitative Genetic Interaction Mapping Using CRISPRI
A. Methods The vectors used in this study were constructed by using standard molecular cloning techniques, including PCR, restriction enzyme digestion and ligation. Custom oligonucleotides were from Integrated DNA Technologies. E. coli strain D1H5a was used for the transformation and selected by 100 μg/ml of carbenicillin, or 50 μg/ml of Kanamycin. DNA was extracted and purified using Plasmid Mini or Midi Kits (Macherey-Nagel). Sequences of the vector constructs were verified with Quintarabio's DNA sequencing service.
Construct Design
The dCas9-KRAB plasmid and sgRNA expressing plasmid are previously described vectors (Du, D. & Qi, L S. Cold Spring Harbor Protocols 2016, (2016)). The SpeI and Sail sites were mutated in the sgRNA expression plasmid. The single sgRNA expression plasmids were cloned as described previously with minor modifications. Briefly, the plasmids were cloned by PCR from an existing sgRNA template using a unique 50 primer containing the desired protospacer (N is the protospacer) and a common primer with (SpeI and SalI sites). The PCR products and the lentiviral mice 16 (mU6) based sgRNA expression vector were digested with BstXI and XhoI and the two pieces of DNA were ligated together. The single vector was introduced unique SpeI and SalI sites to enable the insertion of the mU6-sgRNA expression cassettes.
To construct a lentiviral vector for mU6-driven expression of combinatorial gRNAs, mU6-sgRNA expression cassettes were prepared from digestion of the storage vector with XbaI and XhoI enzymes, and inserted into the target single sgRNA expression vector backbone, using ligation via the compatible sticky ends generated by digestion of the target single sgRNA expression vector with SpeI and SalI enzymes.
The Single Library Cloning
A library of 336 sgRNAs targeting a set of 112 genes encoding epigenetic regulators (3 sgRNAs/gene) was constructed using top prediction hits from the CRISPR-ERA algorithm (Liu, H, et al Bioinformatics 31, 3676-3678 (2015)). The library also included 30 non-targeting negative control sgRNAs. sgRNAs containing XbaI, XhoI, SpeI, and SalI restriction sites, which were used for double sgRNA library construction, were excluded. Individual oligos encoding sgRNAs were synthesized in a 384-well format, pooled, and the single sgRNA expression vectors were constructed individually by ligating the oligos into a common sgRNA lentiviral vector with SpeI and SalI sites. After sequencing validation, 336 sgRNA constructs were manually mixed with equal amount for the single sgRNA screens and double sgRNA library construction. The sgRNA sequence and corresponding genes are listed in Table 5.
Combinatorial sgRNA Library Pool
To generate the pooled storage vector library, the 336 single sgRNA expression vectors were mixed equally. Pooled lentiviral vector libraries harboring combinatorial gRNA(s) were constructed with the same strategy as for the generation of combinatorial sgRNA constructs described above, except that the assembly was performed with pooled inserts and vectors, instead of individual ones. Briefly, the pooled mU6-sgRNA inserts were generated by a single-pot digestion of the pooled storage vector library with XbaI and XhoI. The destination lentiviral vectors were digested with SpeI and SalI. The digested inserts and vectors were ligated via their compatible ends (i.e., XbaI+SalI & XhoI+SpeI) to create the pooled double sgRNA library (336×336=112,896 total combinations) in the lentiviral vector. The lentiviral sgRNA library pools were prepared in DHS ultra-competent cells (Agilent Technologies) and purified by Plasmid Midi Kit (Macherey-Nagel). The sequences of the deep sequencing is listed in Table 6.
Cell Culture
1HEK293T and HEK293 cells were cultured in DMEM supplemented with 10%/6 fetal bovine serum, 100 units/ml streptomycin and 100 mg/ml penicillin at 3TC, with 5% CO2. To generate inducible CRISPRi HEK293 (TetOn-dCas9-KRAB) cell line, the cells were lentivirally transduced with constructs that express dCas9-KRAB from the TRE3G promoter and rtTA. Pure polyclonal populations of CRISPRi cell line were treated with doxycycline, and sorted by flow cytometry using a BD FACS Aria2 for mCherry expression. These cells were then grown in the absence of doxycycline until mCherry fluorescence reduced to uninduced levels.
Lentivirus Production and Transduction
Lentiviruses were produced and packaged in HEK293T cells as described previously with minor modification (Du et al., 2016, supra). Briefly, HEK 293′T were transfected with standard packaging vectors using Mirus TransIT-LT1 transfection reagent (Mirus MIR 2300) according to the manufacturer's instructions. Viral supernatant was harvested 48-72 h following transfection and either filtered through a 0.45 μm syringe filter or snap-frozen.
Growth Competition Assay
Cells were grown at minimum library coverage of 1,000 for the screens. The target cells were infected in the presence of 8 μg/ml polybrene (Sigma) at a multiplicity of infection of about 0.3 to ensure single copy integration in most cells, which is corresponded to an infection efficiency of 30-40%. For single library screens, cells were grown in the flasks and harvested at 0, 12 and 20 days after puromycin selection; for double library screens, cells were grown in the flasks and harvested at 0, 8 and 16 days after puromycin selection. Cells were maintained at least 1,000 cells per sgRNA for each screen.
After the cell samples were collected, the genomic DNA was isolated using QIAamp DNA Blood Maxi Kit (Qiagen) according to the manufacturer's protocol, the cassette encoding the sgRNA was amplified by PCR, and relative sgRNA abundance was determined by next generation sequencing on an Illumina Miseq for single screens or an lllumina HiSeq-2500 for double screens using custom primers with previously described protocols at high coverage (Bassik, M. C. et al. Cell 152, 909-922(2013); Roguev, A. et al. Nat. Methods 10, 432-437 (2013)). Two biological replicates of each screen were performed.
For the cell growth validation experiments, the viruses with single sgRNAs or double sgRNA were transduced into HEK293 (TetOn-dCas9-KRAB) cells, followed by the selection with 2 μg/ml puromycin to remove the uninfected cells. Three days after the cells were treated with or without Dox (0.5 ug/ml), the cell viability was measured by XTT assay (Biotium) according to the manufacturer's experimental protocol. 2,000 to 10,000 cells were plated into 96-well tissue culture plates for the growth assay. For each 96 well, 30 μl of XTT solution was added to the 100 ul cell cultures at the time points indicated. Cells were incubated for 6 hours at 37 C with 5% CO2. Measure the absorbance signal of the samples with a spectrophotometer at a wavelength of 450-500 nm. Measure background absorbance at a wavelength between 630-690 nm. The normalized absorbance values were obtained by subtracting background absorbance from signal absorbance.
Validation of Gene Hits
Cells were harvested and total RNA was isolated using the RNAeasy Kit (Qiagen), according to manufacturer's instructions. RNA was converted to cDNA using iScript™ cDNA Synthesis Kit according to manufacturer's instructions (Bio-rad). Quantitative PCR reactions were prepared with a 2× master mix according to the manufacturer's instructions (Bio-rad). Reactions were run on CFX96 Touch™ Real-Time PCR Detection System (Bio-rad). Primer sequences for qPCR are listed in Table 3.
Results To develop a CRISPRi combinatorial screening approach, a single library consisting of 336 sgRNAs using was constructed using a computational algorithm (Liu, H. et al. Bioinformatics 31, 3676-3678 (2015)), which sequence-specifically targeted 112 genes (3 sgRNA/gene) involved in chromatin regulation (for the gene list and their sgRNAs, see Table 5). The library also included 30 negative control sgRNAs without target sites in the human genome. Pooled cloning of 336 sgRNAs onto itself generated a mixed double sgRNA library containing 112,896 (336×336) combinations. Both libraries were prepared as lentivirus pools ready for large-scale mammalian cell transduction at a low multiplicity of infection (MOI=0.3).
The repressive dCas9-KRAB protein was conditionally expressed under the control of the Doxycycline (Dox)-inducible promoter TetON-3G in the human embryonic kidney 293 (HEK293) cells. Transducing both libraries into clonal HEK293-dCas9-KRAB cells generated two pooled cell populations (FIG. 16A): one with 336 single perturbations and the other with 112,896 double perturbations. Adding Dox to cells could induce expression of dCas9-KRAB to repress target gene(s) guided by co-expressed sgRNA(s) and monitored the growth phenotype from single or double gene perturbations. Pair-ended deep sequencing of sgRNA library distribution for each library (Mi-seq for single library and Hi-seq for double library) was performed with and without Dox, as well as at different time points.
It was first investigated if sgRNA distribution remained consistent between biological replicates before and after library screening. Sequencing single and double libraries with or without Dox at different time points exhibited consistently high coefficient of determination (R2) (FIG. 15B-E). For example, R2 was 0.980 without Dox induction and 0.971 with Dox for the single library (day 20) (FIG. 15B-C); and for the double library (day 16), 0.934 without Dox and 0.906 with Dox (FIG. 15D-E). sgRNA distribution from biological replicates was assayed at other time points and similarly high correlation was observed. Together these data demonstrate that the experimental platform produces data of very high reproducibility.
It was next determined if inducible expression of dCas9-KRAB allowed one to identify single and double gene perturbations that influenced cell growth (FIGS. 15F & G). It was observed that repression of a set of individual genes dramatically slowed down cell growth in the presence of Dox compared to without (FIG. 15F). This list of genes included gene components of the mediator complex (MED14 and MED15), components of the histone H3-Lys4 methyltransferase complex (WDR82 and WDR5), and RNA polymerase II associated factors (PAF1 and RTF1). Double library culture showed a large number of combinatorial perturbations significantly reduced cell growth with Dox, with an overall bifurcation pattern, wherein the negative controls fell along the diagonal line and the positive controls were biased from the diagonal line (FIG. 15G).
The above inducible experiments were performed at end time points. sgRNA distribution was further compared for both single and double libraries with and without Dox induction at intermediate time points (day 12 for single library and day 8 for double library). Consistent phenotypes at these time points compared to end time points were observed. For example, a similar list of genes whose repression slowed down cell growth, including ME14, MED15, WDR82, PAF1, and RIF1. The absence of WDR5 at day 12 indicates that WDR5 has a moderate role for growth compared to other gene hits. For the double library, a similar bifurcation pattern was observed, with a difference that the bifurcation degree (measured by the angle between the two populations) is smaller at earlier time points.
The consistent gene hits and dropout pattern for both libraries between different time points propelled a comparison of datasets across a broad time course. It was investigated if the trend of dropout effects could provide another layer of identification of true positive hits (FIG. 16). For the single library in the presence of Dox, sgRNA enrichment was compared at days 0, 3, 7, and 13 (FIG. 16A). While some genes showed consistent depletion (e.g., RTF1, MED14, SAP30), many other genes showed inconsistent enrichment (e.g., MRGBP). Among 112 epigenetic factors, 20 genes were observed to exhibit consistent depletion over time, showing inhibition of these genes constantly slowed down cell growth (FIG. 168). The double library similarly showed temporal dropout of pairwise sgRNAs assayed at days 0, 8 and 16 (FIG. 16C). Over time, a large number of combinations were consistently depleted as a selection of these was plotted as in FIG. 16D.
The time-course sgRNA enrichment was compared in the absence of Dox for both single and double libraries. No significant changes of sgRNA distribution were observed over time for both libraries without Dox. For the single library, comparing the day 0 sample with day 12 or day 20 samples (+/− Dox) showed only dropout of gene hits with Dox (FIGS. 17A-B), and for the double library comparing day 0 with day 8 or day 16 (+/− Dox) confirmed similar conclusions. Altogether, these experiments confirmed that the system enables inducible, temporal screens of genetic interactions.
Two negative interactions were validated, demonstrating their ability to suppress cell proliferation and causing repression of target endogenous genes. Two pairs were chosen for testing: MGBRP/MED6, and BRD7/LEO1. MRGBP is a component of the NuA4 histone acetyltransferase complex involved in gene activation by acetylation of histones; BRD7 is a member of the bromodomain-containing protein family; and LEO1 is a component of the PAF1 complex (PAF1C) involved in transcription of RNA Pol II. The results confirmed the validity of the double repression and synthetic lethality-based growth effects. As shown in FIGS. 16E & 16F, repression of two genes simultaneously (MGBRP & MED6 for FIG. 16E; and BRD7 & LEO1 for FIG. 16F) suppressed cell growth over time, while repression of individual genes did not cause significant growth inhibition. Quantitative PCR results further confirmed the repressive effects of the sgRNAs on the corresponding genes either individually or combinatorically delivered into cells. Notable, the moderate repression effects of the tested sgRNAs supported the strong growth effects of the genetic interacting pairs. Future optimization of CRISPRi repression efficacy allow one to perform screens at different strengths (weak, medium, strong) of gene repression.
Based on the curated set of protein complexes and pathways, a GI map depicting the genetic cross-talk between different functional modules involved in chromatin was created (FIG. 17). Using a scoring system similar to the S-score (Collins, et al., J. Meth. Enzymol. 470, 205-231 (2010)), 68 negative and 47 positive genetic interactions were identified. Contained within this map are modules corresponding to the INO80 chromatin remodeling complex; the mediator complex (MED); the NuA4 histone acetyltransferase (HAT) complexes; the Nucleosome Remodeling Deacetylase NURD complex; the histone methyltransferase (HMT) complex SET1A/B; the Polycomb complex PRC1; the histone 3 lysine-4 methyltransferases MLL3/4; the SIN3 transcription repressor; Host Cell Factor C (HCFC)-glycosyltransferase (OGT) complex; and nuclear THO transcription elongation complex. Notably, the mediator complex occupies a large set of interactions on the map, interacting strongly, both positively and negatively, with many other functional modules. For example, strong positive GIs were observed between the MED complex and modules corresponding to PRC1 and the SET1A/B complex. Furthermore, strong negative interactions were observed between components of the SIN3 complex and many other modules of mediator components and SWI/SNF family of protein SMARCC2.
The nuclease Cas9 for gene editing-mediated knockout allows complete loss of function, yet knockout can be heterogeneous among alleles due to existence of in-frame indels. On the contrary, CRISPRi-based dCas9 transcription knockdown leads to partial, homogeneous loss of function (Mandegar, M. A. et al. Cell Stem Cell 18, 541-553 (2016)). Applying the two methods to higher-order genetic screening needs to consider these important differences. For example, as epistatic genetic screens require simultaneous perturbation of multiple genes (usually 2 genes, 4 alleles), the heterogeneity of gene knockout in pooled CRISPR screens may result in a significant portion of cells without proper epistatic perturbation. Among the cells that are properly perturbed, complete knockout of function offers a highly sensitive way to discover novel gene combinations whose perturbation leads to measurable phenotypes (e.g., growth). Yet, combinatorial multiple gene knockouts may easily cause lethal effects by itself, precluding testing other phenotypes (e.g., differentiation or host-pathogen interaction).
On the contrary, partial knockdown by CRISPRi, while being less sensitive than CRISPR knockout, likely avoids major dominating lethal effects. The homogeneous transcriptional repression could generate cell populations with consistent multi-gene perturbation. Furthermore, sgRNAs binding at various loci along the promoter lead to varying levels of CRISPRi repression, which is contemplated to provide dosage-dependent combinatorial screening distinct from binary perturbation from CRISPR. The demonstration of the inducible and titratable features of CRISPRi combinatorial screening showed the method allows assaying genetic interactions temporally and potentially in a dose-dependent manner.
Compared to RNAi-based methods, the major approach for genetic interaction mapping, CRISPRi presents a few advantages as well. CRISPRi knockdown is specific (Gilbert, L. A. et al., Cell 159, 647-661 (2014)), with less concerns about multiple sgRNAs in the same cells causing unexpected off-target perturbation. As CRISPR activation (CRISPRa) is based on somewhat similar setup as CRISPRi, by changing a repressive effector into an activating effector, the same approach can be expanded into gain-of-function screening of pairwise of genes. Furthermore, combining CRISPRi and CRISPRa into the some cells is contemplated to allow simultaneous activating a gene while repressing another gene. These dramatically expand the modes of epistatic screens that can be performed.
Development of high-throughput epistasis-mapping technologies has made it possible to interrogate complex biological phenomena. Mapping the PPI networks and GI networks have become major methodologies to study epistasis. The PPI networks report on gene products that interact physically; (GIs, in contrast, illustrate functional relationships between genes including, but not limited to, physical interactions of their gene products. They often reveal how groups of proteins and complexes work together to carry out biological functions and can describe the cross-talk between pathways and processes. Therefore, the method for mapping GI networks in mammalian cells provides a useful, natural complement to PPI mapping methods and other existing GI mapping methods. Integrating the two types of information is extremely powerful in understanding complex biology in broader contexts of basic and translational research.
TABLE 5
Gene list and sgRNA sequences
Gene name sgRNA sequence SEQ ID NO
ACTL6A_1 GTGGGTGGCGGTGGAAGTTA 1
ACTL6A_2 GGCCGCGACTGCGAGTCTCG 2
ACTL6A_3 GCGCCGGCAGCAGCCATGAG 3
ACTR8_1 GCGCTGCAGCCACGACTGCC 4
ACTR8_2 GTCTCCGGCCATAATGACCC 5
ACTR8_3 GCGGCCCATCGTGCCCGCGC 6
ARID1A_1 GGCTCTGTAGGCTCGGGACC 7
ARID1A_2 GGAGAAGACGAAGACAGGGC 8
ARID1A_3 GCCCCCCTCATTCCCAGGCA 9
ARID1B_1 GCATCCTCTTCCTCCTCGTC 10
ARID1B_2 GGGGAGCAGCCCCGTCTCCA 11
ARID1B_3 GAGGCGGCTCTCAAGGAGGG 12
ARID2_1 GGAACTGCCGCAGCTCGTCC 13
ARID2_2 GAACCGGGGGGGCAGCGCCG 14
ARID2_3 GGGGTCCCGGCTGACAAGTG 15
ASH2L_1 GGAGCGGTCGCAAATGCAAC 16
ASH2L_2 GCAGCCGCTCCTCCTGGAGA 17
ASH2L_3 GTGGCCGTGATGGCGGCGGC 18
BRD7_1 GTCGGACAAACACCTCTACG 19
BRD7_2 GGGCTTCCGCTCTTTCCCAG 20
BRD7_3 GCAGGCCCAGGCCGGCGAAG 21
BRD9_1 GCTGGCACCCGGTCGGACCT 22
BRD9_2 GAGTGGCGCTCGTCCTACGA 23
BRD9_3 GCGAGCGCGGGCGGCCAGCC 24
CBX2_1 GTACTCCAGCTTGCCCTGCG 25
CBX2_2 GCTGAGCAGCGTGGGCGAGC 26
CBX6_1 GTGGGTGCCGCTGAGCAAGA 27
CBX6_2 GCTGTCTGCAGTGGGCGAGC 28
CBX6_3 GCATCGAGTACCTGGTGAAA 29
CBX7_1 GCTGTCAGCCATCGGCGAGC 30
CBX7_2 GTGCGGAAGGTGAGGCTGCC 31
CBX7_3 GCACCGCTCCCTCCACGCTG 32
CBX8_1 GCTCCTGGAAGCGGCCAAGG 33
CBX8_2 GGTGGGGGAGCGGGTGTTCG 34
CBX8_3 GCACGGAGGCCCTAGGCCCG 35
CHD3_1 GCTCCCACTCGGGCTTGGGG 36
CHD3_2 GTCTGCCGCCTTCATCACAC 37
CHD3_3 GAGGAAAAGAAATCCTCAGC 38
CHD3_4 GTTTTAGGCTACTTGGGAGG 39
CHD4_1 GCTCCGGCTCCTCCTCGCCG 40
CHD4_2 GCGCGACCTGCGGCGGCTCC 41
CHD4_3 GGCCGTGAGGGGCGTCTCTT 42
CNOT1_1 GTCGAGGAGAGCCGGAGTCG 43
CNOT1_2 GGAGCCGCCTGAGGTGAGGC 44
CNOT1_3 GTTTCTCTACAAAATGGCGC 45
CNOT2_1 GAGCCTAGGGGAGTGGAGTC 46
CNOT2_2 GCCGCCTTCTCTTCTCCCCC 47
CNOT2_3 GCAGCTCCAGATCCTAGGCC 48
CNOT3_1 GTCAGCTTCCGCGGAGCCAT 49
CNOT3_2 GTTGTTCTGACGACGGGGGT 50
CNOT3_3 GCCGCTATCGCGATAGCGCC 51
CTR9_1 GTGAGTGACGGCTCCGGCTC 52
CTR9_2 GGAGACTACCGGCTGCGGAG 53
CTR9_3 GATGGAGCCCCGCGACATGA 54
CXXC1_1 GAATGAATACAACTTGATCC 55
CXXC1_2 GAACCTCTCTGCCTGACAAA 56
CXXC1_3 GGACGGCTGTGTGCCTTGCG 57
DMAP1_2 GGCCGTTAGGAACATCCAAG 58
DMAP1_2 GCGGGCCAAGAGGAGAAGGG 59
DMAP1_3 GACCCAGGTGCGGAAGTGCG 60
DPY30_1 GAGTGGGACAGTCCACGACT 61
DPY30_2 GTGCTCCCGCGCCCAGGTGG 62
DPY30_3 GATTTCAACACGAAGACTCC 63
EED_1 GAGAAGAGGCGAAACTCAAA 64
EED_2 GCTGAAACGTCTTTGGAAGG 65
EED_3 GTAAGGTCCGTTGGATTAAG 66
ELP2_1 GGACTCCCCGCACCCGGTTT 67
ELP2_2 GTCATAGAGCACCACGGAGC 68
ELP2_3 GGTGCCACCATGTCGCCAAC 69
ELP3_1 GAAGCGGAAAGGTGCGAAAG 70
ELP3_2 GCCTGGGCGTTCGCCCCTTT 71
ELP3_3 GCAGCCACAAACTCAGACCA 72
ELP4_1 GCCAGCGTGACCAACGACAG 73
ELP4_2 GGTAGTGTTGCCGCGAGTAC 74
ELP4_3 GCAACGTCACCAGTTTCCAG 75
EP400_1 GCGTCAGGAGGGCGGGAGGA 76
EP400_2 GGTAAGTGAGGGCGGAGGCG 77
EP400_3 GGCTACGCGACCCCGGACCC 78
EPC1_1 GGCACTAACACCAGCCGGGA 79
EPC1_2 GCTGCCGGGGACTTGAGGGG 80
EPC1_3 GTTGGCTGAAGAGCGCACAG 81
EZH1_1 GTGAGTAAACAAGCCTGGGC 82
EZH1_2 GGAAATTGGAAGGAATCCGA 83
EZH1_3 GGCGCCCCTCCTCATTCCGA 84
EZH2_1 GGATTTCGGGGTGCGTCGTG 85
EZH2_2 GCTGCCCTCGCCGCCTGGTC 86
EZH2_3 GGGGATGTACACAATGAAGT 87
HCFC1_1 GAAAGGAGCAACAAGCGCCG 88
HCFC1_2 GGGCTACGACTGAGGAAGGG 89
HDAC1_1 GGGACGGGAGGCGAGCAAGA 90
HDAC1_2 GGCTGAGGCTGGAGCGCCGA 91
HDAC1_3 GCTCGGAGAGGAGGCTGCGA 92
HDAC2_1 GGCTCGGTACCACCCGGCAG 93
HDAC2_2 GGCGATAGTCCCGCGGGGAA 94
HDAC2_3 GGCACCAACTCGCGAGGAGG 95
IKBKAP_1 GTTTGGGCAGATGGGCAAGA 96
IKBKAP_2 GCCTGGCACCGTAGAGGTAG 97
IKBKAP_3 GGCGAGGCCGGGCCCGCTTC 98
ING3_1 GAGGGAACAAGGGGGTCCAG 99
ING3_2 GGAAAGTGAGTGCGCGGCGC 100
ING3_3 GAGTTTTGTCCCCTCCAATA 101
INO80_1 GGGGTCCCAGGAGCCGCGGA 102
INO80_2 GGTTCGCTCTCTGAGGCCGT 103
INO80B_1 GAAAGGGGACTAGAAATGGT 104
INO80B_2 GCGGCGTGGGAGCACCTCTG 105
INO80B_3 GCGAATAGATCAAGCAATTT 106
INO80C_1 GAAGACTCGGAGTGCGATGG 107
INO80C_2 GTTCCGGACTATTCCGGGAG 108
INO80C_3 GGAAGTTCCAAGGCCCGCGC 109
INO80D_1 GGCTGACAGATCAGAGTGAG 110
INO80D_2 GGAGCCCGGGGATGTGGGCC 111
INO80E_1 GGTAGCGGGAGGGCAGACTC 112
INO80E_2 GTCATGAACGGGCCGGCGGA 113
INO80E_3 GTGCTGCCGCGGGAAGGCTG 114
JARID2_1 GACTCGGCGAGCCCTCGCTG 115
JARID2_2 GTTACATCTTGGAAAAGAAA 116
JARID2_3 GGGGGGGGAGTGAAGGGCGT 117
KAT5_1 GCAAGACTGCCCCTGTGACT 118
KAT5_2 GCCTCACGAAGCCCCTGTAG 119
KAT5_3 GCCACTGGCTGTGCACGTTA 120
KDM1A_1 GACAGAGCGAGCGGCCCCTA 121
KDM1A_2 GGCGGCCCGAGATGTTATCT 122
KDM1A_3 GCGTGAAGCGAGGCGAGGCA 123
KDM2B_1 GCTCGGCTTCCATACCTATA 124
KDM2B_2 GCGGACCCGCCATGTGGAGG 125
KDM2B_3 GTCGGCCACACAGGTAATGT 126
KDM6A_1 GCAGCCACAGGCGGGGACGG 127
KDM6A_2 GAAAGCCGCCGCTGCCGACC 128
KDM6A_3 GGAGCACTGAGGGGATTCGT 129
KMT2A_1 GAGGCGGCGGCCGCTCCCCC 130
KMT2A_2 GGCCGGCCCTGAAGAGGCTG 131
KMT2A_3 GGCGCTTCCCCGCCCGACCC 132
KMT2D_1 GATAAAGATTCAGAACCGGC 133
KMT2D_2 GTGCCAGGACCAGAAATGTA 134
KMT2D_3 GAGATTATCCAAAACCTGAG 135
LEO1_1 GTGAGCGATAATGGCGGATA 136
LEO1_2 GCGAAGCTGAGCGTAAAGGT 137
LEO1_3 GCGTGGCAGGCCTTCCGCTG 138
MBD2_1 GGATTCCAAGGGCTCGGTTA 139
MBD2_2 GGGCTGGATGCGCGCGCACC 140
MBD2_3 GGACCTAAGAGGCGGTGGCC 141
MBD3_1 GGAAGAAGTGCCCAGAAGGT 142
MBD3_2 GAGCCCGTTGAGGCCCTGCG 143
MBD3_3 GCGCAATGGAGCGGAAGAGG 144
MED1_1 GATCAATCTGAAGTCCCCGG 145
MED1_2 GGCTCGGGATCCCGGGACGC 146
MED1_3 GAAGCTAGATCCGCCACAAA 147
MED10_1 GGAGAAGTTTGACCACCTAG 148
MED10_2 GTTGAGCCCGGCCTGGCTGC 149
MED10_3 GGTCTCCCCAGGGCCTGGCC 150
MED12_1 GCGGCCGAGAGACAACAAGG 151
MED12_2 GAGGGAGCCGAAAAGGGGGG 152
MED12_3 GTAGCGCCGGAGGCACCAGC 153
MED13_1 GCCGGCGGCGGCTGCTGTGA 154
MED13_2 GGTTACAGTGACAATCTTCC 155
MED13_3 GGTGCGCCCTTGGGCCGTGG 156
MED14_1 GACTCTGCCCGCTCCCGTTT 157
MED14_2 GTGTGCCGTTGCGCCAAGCC 158
MED14_3 GTGGTTCTCCAGCTGCACTG 159
MED15_1 GATACGGGCGGCGGGAGCTG 160
MED15_2 GGTCAGTCAAATGTGAGTAG 161
MED15_3 GCCGCCTCAGTCACAGAGCC 162
MED17_1 GGGAGCTTGCGGTGCGTTCT 163
MED17_2 GCGTTGCGTTCGGTTTCCCG 164
MED17_3 GAGGCTTCCCTGCGGAGAGC 165
MED23_1 GGAATATAGGGGCAGAGGGG 166
MED23_2 GGCGGGGGTGATAGTACAGA 167
MED26_1 GGCGGCTCCTCCTCCTCCTT 168
MED26_2 GTCACTCACTCGCCGGCCTC 169
MED26_3 GGCGTCTCCGCAGCAGATCA 170
MED4_1 GCGGCTGCTGTCTGCGCTTG 171
MED4_2 GGCGAGCCTGAGAGCCGGGC 172
MED4_3 GGAGCGGCTGGGAGGCGGTT 173
MED6_1 GTTTCGCTAGATCACAGCCT 174
MED6_2 GATTGTCTGTGGACCAGTTT 175
MED6_3 GCGTTTACAGGTTCTCTTTC 176
MED7_1 GAAAGACGAAAGACCGCCTT 177
MED7_2 GTGCGGTCTCTCCGAGAGCG 178
MED7_3 GGCTCTAAGCGTGGCAGTCT 179
MED8_1 GACCGAGAGTGGGCTGGCTA 180
MED8_2 GGCAGAACCCACGGCTGATA 181
MED8_3 GCGTTGGGCGTACTAGCGGC 182
MEN1_1 GTGGGATGTAAGCGCGGAGG 183
MEN1_2 GACAGACTTTACAGCCCCGG 184
MEN1_3 GGACTCTCCTTGGGGTTTGG 185
MRGBP_1 GCTCGGCCGGGCCGCGGCCA 186
MRGBP_2 GCCGCAGGCGACAAGGGCCC 187
MRGBP_3 GACAGTGGTGTGGAGCCCCG 188
MTA1_1 GCCGCCAACATGTACAGGGT 189
MTA2_1 GTTGGGCTCTGCCGGCCGCA 190
MTA2_2 GAACGAGCTCGGCTCCTGCC 191
MTA2_3 GCCTCAGCGTCCCGGAGTG 192
MTA3_1 GCCCCAGAACGTGGGGGCCG 193
MTA3_2 GTCCAGGCGCGCTACACGTT 194
MTA3_3 GGGGAGGAACGCCTTGTCAC 195
NCOA6_1 GTCGGGCTGGCTTCGCGGGG 196
NCOA6_2 GACCGTGCCACTCGGTCGCC 197
NCOA6_3 GACGGCGGCGCGGGCCCGTA 198
OGT_1 GCTCTGGAGGGCTTGAGCGG 199
OGT_2 GCTCCAGATGGCGTCTTCCG 200
OGT_3 GATGGTCAATTAGAGTTCCC 201
PAF1_1 GTGAACGCGCAGGCAGCACC 202
PAF1_2 GCGGAAAGTGGGTTGAGATG 203
PAF1_3 GCGGCCTGAGGAGACCCGTT 204
PBRM1_1 GGGTAAGGCCGGGCCCAGGG 205
PBRM1_2 GGCCCGGCAGCTGACCAAGG 206
PBRM1_3 GCAGGTGCGACAAGGCTACT 207
PCGF1_1 GCCTCATCGCGATCGCAATC 208
PCGF1_2 GATGGACCCGCTACGGAACG 209
PCGF1_3 GTCGGCCAGCGGTGCGAATT 210
PCGF2_1 GCTTACCTGGGTTCGGGGTC 211
PCGF2_2 GCCTGTAACCCTCTGGGGAT 212
PCGF2_3 GGGGGGTGCGAAGGCAGGAT 213
PCGF6_1 GTAGGCGCTGCCAAAACCGA 214
PCGF6_2 GGCGCCTCTGTCTGAGACGG 215
PCGF6_3 GGTGTCTCTCCCGACCATGG 216
PHC1_1 GAAGGTAACCGGGCGACCGA 217
PHC1_2 GGGCGTTACACAGATGGAGG 218
PHC2_3 GCTCAGCGCCGGAGGTAGGC 219
PHC2_1 GACTGGCAGCTCATTCTCCA 220
PHC2_2 GTACACAGAAATCTGGGGCC 221
PHC2_3 GGTAAGAGTCTAATTGATCT 222
PHC3_1 GTGACTGATGTCGTAACTAG 223
PHF10_1 GGGCCCACGCCCCGGCACCC 224
PHF10_2 GTCGCTGTCGCACGGCCGCG 225
RBBP4_1 GGCACCCTCACCTTCCTTGT 226
RBBP4_2 GCTGAGCCGCGGCCTCGACA 227
RBBP4_3 GGGGGCGCAGGAAACAATAG 228
RBBP5_1 GTTGTTGCCGGAGCTGAGAC 229
RBBP5_2 GCTGCGTTTTAGAGAAGCGT 230
RBBP5_3 GGTGGACGCCGCGAAGAGAC 231
RBBP7_1 GGAGCGCAGCCGCTGGAGGA 232
RBBP7_2 GCGCGCGCGTTGACCGCCTC 233
RBBP7_3 GCCCTTGTCCGGGGGTTGCT 234
RTF1_1 GGCGGGCAAGAGGGGAGTCC 235
RTF1_2 GGACCACCATGGTAAAGAAG 236
RTF1_3 GCGCGGGCCGGCGGAGCCAG 237
RUVBL1_1 GGGCGCACTGTCCTAGCTGC 238
RUVBL1_2 GCCTCCCACAGCCACGTGAA 239
RUVBL1_3 GCAGGCGGCCTCAGGGCTTG 240
SAP18_1 GGTCAGGGCGAGCGTCTCGC 241
SAP18_2 GGAGTCGCGCGTTACCCAGG 242
SAP18_3 GATCGACCGCGAGAAGGTGA 243
SAP30_1 GTGAGCGGGGTCCCCGCTCC 244
SAP30_2 GGCCCGGGACAGTTGGTGTT 245
SAP30_3 GCAGAGTGAATTGCCGCTGC 246
SETD1A_1 GAATAGCCCGCTTCTGTCCC 247
SETD1A_2 GCCAGCAGGGATTGGCTAAC 248
SETD1A_3 GACTCCACCAAGGCGGATGA 249
SETD1B_1 GGTTCCTCCTCTCGCCCGAA 250
SETD1B_2 GATTGACCCGGCTCTGAAAA 251
SETD1B_3 GCACGGCTGGGGGGGCGCGC 252
SIN3A_1 GGGCTAGTCCGCCGGCCGCT 253
SIN3A_2 GCTCGGTCCCAGGGCCCGCA 254
SIN3A_3 GGCCTGTCCCTCGCCTACCT 255
SIN3A_4 GCGGCCGCTTCTCTGTTACC 256
SIN3A_5 GCCTGTGACCGCTTCGTTAG 257
SIN3B_1 GGGACGCCACTCACGTGCAC 258
SIN3B_2 GAGGGCCGAGGTGAGAGGTG 259
SMARCA4_1 GGGCGGTTTGAATGGAGCCG 260
SMARCA4_2 GGCGCGCCCTGTGCGGGGCC 261
SMARCA4_3 GGGAAGGCCACAGTGTCGCG 262
SMARCB1_1 GGCCTGGTCGTCGTCTGCGG 263
SMARCB1_2 GGGCCGAGGGAAACCGAAGC 264
SMARCB1_3 GCGAGGGATCAGGAGGGCTG 265
SMARCC1_1 GCTGTTTATCGACGGAAGGA 266
SMARCC1_2 GACGGTGTCCCAGCTGGATT 267
SMARCC1_3 GGTGGGTTCGCGCGCCCGTG 268
SMARCC2_1 GACAACGTGCGGCTGTGGCT 269
SMARCC2_2 GACCGCGGCCCTGCAGCCCC 270
SMARCC2_3 GCCTCGTAGTACTTCACGTT 271
SMARCD1_1 GTGGCTCCAAGCGGCGGCGC 272
SMARCD1_2 GCCGCACAAAGAACCGGAAC 273
SMARCD2_1 GACTCGGGCGGCCAAACCTC 274
SMARCD2_2 GCCCGGGAGATTCCGGATCC 275
SMARCD2_3 GGAACTCGCGAACTTGGATT 276
SMARCD3_1 GAATGGGAGTCTGCCAGTCA 277
SMARCD3_2 GCCAGGCAGCGATGGGGAGG 278
SMARCD3_3 GAAAGTGCTCGGCAGGGGGG 279
SMARCE1_1 GCGGGTGAGTGTTTCCAAGT 280
SMARCE1_2 GAACTCGGGGTCTAGCCAAG 281
SMARCE1_3 GGCCTCAAGGAGGCCTCAAC 282
SRCAP_1 GTCAGTCCGTCGGGAGGGCT 283
SRCAP_2 GCTCGGGTCTTGGGAACGTG 284
SRCAP_3 GTGTGAACCCGCAGGAGGCC 285
SUZ12_1 GGGCGAGCGGTTGGTATTGC 286
SUZ12_2 GGCGGGTAGCTGGCGGGGGG 287
SUZ12_3 GCCTCAGAAGCACGGCGGTG 288
THAP1_1 GTGATGGTGGCCTCCCTCGG 289
THAP1_2 GTTCTCAGTGTCGCTGCGCT 290
THAP1_3 GCTAATGCAAACAACAAAAC 291
THAP3_1 GCTGCCCCCAACAAAGATGG 292
THAP3_2 GGGTCCCCGCCTCTTACCGG 293
THAP3_3 GGGCCCGCGGACCGACTCCG 294
THOC1_1 GCTTCGGGCAAACTGAAGAG 295
THOC1_2 GGCAAAATTCGAGTAATTTC 296
THOC1_3 GTCCGCCTCAGCGTCCGCTC 297
THOC2_1 GAGGCGAATTGTGAGTGTTC 298
THOC2_2 GCTGCACTCTCACCTGTAGT 299
THOC2_3 GACCATCCACGCCCGCCGCC 300
THOC3_1 GCTGCTGCAGTGTTGTGAGT 301
THOC3_2 GGCGGTCCCCGCTGCAGCCA 302
THOC3_3 GCCCCGGCTCGATGGCCCCG 303
TRRAP_1 GGGTCGCGGGCCGGGCCTGC 304
TRRAP_2 GGCGGGCGTCCGAACGGCCC 305
TRRAP_3 GCGGCCGAGCGGTTGCGACG 306
WDR5_1 GGCCGCACAGGAGACAAGGG 307
WDR5_2 GCTCTGGCGGCCTCGGTCTC 308
WDR5_3 GGCACGCACCTTGCTCTGAG 309
WDR82_1 GAGGTGGCTGTGAGGACGAA 310
WDR82_2 GGAGGAGGCGGCCCAACTGT 311
WDR82_3 GCGGAGCTTCCGCGTCGCTA 312
DC13_1 GGGCTGAACGCGTATTCGCG 313
DC14_1 GGCGATTCGGCGACCTTAGT 314
DC14_2 GGCGCGAGTACGAAATTAAT 315
DC14_3 GATTATCAGACGCGCTGCGT 316
DC15_1 GCGCGGCTAGAATAGACTTG 317
DC15_2 GGTTCGTGCGGTAGTGTGCG 318
DC15_3 GTATCGTCTTCCGTCCTCGT 319
DC15_4 GGCTACTCTATGCGTCGATT 320
DC15_5 GCTTAACAAAGCGAGCGACC 321
DC15_6 GGCACTGGACGATATCCGAC 322
DC15_7 GTTCATCTCAACGGTAATCG 323
DC1617_1 GGTTATATTGACGTCCTGCC 324
LC_1 GCTAGTCTGCGTGACGCGTCT 325
LC_2 GAAGTAACTGAAGGATCAATAT 326
LC_3 GCGGGAAAACCGCGCCCCGGA 327
LC_4 GCTCAGGGCCGTGACGCGTGGG 328
LC_5 GTAGGAGCGCGTGCTGATTGT 329
LC_6 GGACGAACTAATGTATTGTGGC 330
LC_7 GGTTTATGGACCTTCAGGGAG 331
LC_8 GGCGTACCCGTGGTTTCACCGT 332
LC_9 GCTTGGGAGCAAGCCGGCGGTA 333
LC_10 GTGTGGCGACCCTGGTCTCAT 334
LC_11 GGGCCTCTGTGAGGTCGTGGT 335
LC_12 GTATGATACTCGTGCTTAGT 336
LC_13 GCGAAGTCGAATGTTGGTCG 337
LC_14 GGCCCAACATCCTCGTGTCCA 338
LC_15 GTGGCGGAGCCTAGCCGAGAGT 339
LC_16 GGCGCGAACTTTAAGGTGGAC 340
LC_17 GATTAGTTCGCGTATGGCAGCA 341
LC_18 GCCGTAAGGACGGGTAGAGGT 342
LC_19 GGGGGCGGAAATCGAGCCCT 343
LC_20 GAAGTGAGAGGAGGGAGCAGCC 344
LC_21 GTAAATCCCGGAGTCAGA 345
LC_22 GTGAGCGGCGACCCCCCCTG 346
LC_23 GGTGCGGACCCCCGCCGGGGG 347
LC_24 GGTGAGCCGGTTTGTGAGAAG 348
LC_25 GAGAGTGCGCTGCAATGGATAT 349
LC_26 GGATGTGCCATGGTGAGGGCTG 350
LC_27 GGATGCGCCTAGGCGAAAGAAA 351
LC_28 GAGCCGATGCAGGGCGTAGGG 352
LC_29 GCCATTCTCTATGTTCGATAAG 353
MCM2_PC GGATCGTGGTACTGCTATGG 354
INTS9_PC GGCAGGTGGCGGAGATTGCAC 355
GEMIN5_PC GGCGTGAGGCTACGAGCGGT 356
CENPA_PC GCCAAGCACCGGCTCATGTG 357
POLR1D_PC GGAAGCAAGGACCGACCGA 358
TABLE 6
Regular primers for cloning and sequencing
primers to clone single sgRNA:
Forward GGAGAACCACCTTGTTGGN19GTTTAAGAGCTATG
primer CTGGAAACAGCA (SEQ ID NO: 359)
(N19 is the
targeting
sequence):
Reverse CTAGTACTCGAGNNNNNNNNNNGCGTCGACCCTAG
primer GGCTAGCACTAGTAAAAAAAGCACCGACTCGGTGC
(N10 is the CAC (SEQ IDNO: 360)
barcode
sequence):
PRIMERS TO AMPLIFY GENOMIC DNA FOR SINGLE
SCREENS:
Forward AATGATACGGCGACCACCGAGATCTACACGGTAAT
primer: ACGGTTATCCACGCGG (SEQ ID NO: 361)
Reverse CAAGCAGAAGACGGCATACGAGATNNNNNNNNGCA
primer CAAAAGGAAACTCACCCT (SEQ ID NO: 362)
(NNNNNNNN
is the
index):
CUSTOM PRIMERS FOR M1SEQ:
Read2 GTGTGTTTTGAGACTATAAGTATCCCTTGGAGAAC
primer: CACCTTGTTGG (SEQ ID NO: 363)
Index read GTCTCAAAACACACAATTACTTTACAGTTAGGGTG
primer: AGTTTCCTTTTGTGC (SEQ ID NO: 364)
PRIMERS TO AMPLIFY GENOMIC DNA FOR DOUBLE
SCREENS:
Forward AATGATACGGCGACCACCGAGATCTACACTGAGAC
primer: TATAAGTATCCCTTGGAGA
(SEQ ID NO: 365)
Reverse CAAGCAGAAGACGGCATACGAGATNNNNNNCTGGC
primer GAACTACTTACTCTAGCTTCCCGGCAACGCCTTAT
(NNNNNN TTAAACTTGCTATGCTGT
is the (SEQ ID NO: 366)
index):
CUSTOM PRIMERS FOR H1SEQ-2500:
Read1 CGAAGTTATAAACAGCACAAAAGGAAACTCACCCT
primer: AACTGTAAAGTAATTGTGTG
(SEQ ID NO: 367)
Index read GTTTAAATAAGGCGTTGCCGGGAAGCTAGAGTAAG
primer: TAGTTCGCCAG (SEQ ID NO: 368)
Read2 GCACCGACTCGGTGCCACTTTTTCAAGTTGATAAC
primer: GGAC (SEQ ID NO: 369)
TABLE 7
qPCR primer sequences
Gene
name Forward primer Reverse primer
SIN3B TTACTGCATGTCCAAGTTCAAGA CCAGGTGTCGTTCAGTA
(SEQ ID NO: 370) CCC
(SEQ ID NO: 371)
MED4 GGTGGTAACAGCACACGAGA TTGCCAGCATTTCTATA
(SEQ ID NO: 372) AGTTCC
(SEQ ID NO: 373)
MED6 TGCAGAGGCTAACATTAGAACAC GCTGTTGCTTCCGAATG
(SEQ ID NO: 374) ATGA
(SEQ ID NO: 375)
MRGBP TGAACCGACACTTCCACATGA TGGTCCCAGATGACCTT
(SEQ ID NO: 376) GGAT
(SEQ ID NO: 377)
Example 3 Repression Screening Platform Besides gene activation, gene repression also can facilitate cell fate conversion. For example, knockdown of many epigenetic modulators increases the efficiency of reprogramming or transdifferentiation processes. This example describes, a repression screen platform to identify cell fate conversion barriers genes.
To perform gene repression screens, a clonal mouse ES cell line carrying Staphylococcus aureus (SaCas91-KRAB is co-transfected with Cas9, sgRNA targeting mouse Rosa 26 loci, and a vector containing dCas9-KRAB with a Zeocin-resistance gene. Zeocin-resistant cells are sorted into a 96-well plate. After a week of culture, the genome is purified and the correct integration of SadCas9-KRAB into Rosa 26 loci is confirmed. This clonal cell is used as a platform to identify gene barriers of differentiation processes.
To perform single gene repression screens, a genome-wide gene repression SadCas9 sgRNA library is generated. The library includes sgRNAs targeting −50 bp to +300 bp region relative to all putative genes in the mouse genome. All the available sgRNAs are blasted through mouse genome and excluded if there is predicted off-target binding. Other design criteria and construction method are similar to the design of activation sgRNA library described in Example 1. This repression library is transduced into the SadCas9 repression mouse ES cells, and neural differentiation is performed as in the single screen. On day 12, cells are harvested and sorted for hCD8+ and hCD8−. The sgRNAs are sequenced, paired-analyzed for enriched genes in hCD8+ and hCD8− populations, and a list of top hits for neural differentiation barrier genes is identified.
Over the past years, the literature has shown that the activation of combinatorial transcription factors can control a cell fate. For example, the transcription factors Oct4, Klf4, Sox2, and c-Myc are used to reprogram somatic cells to induced pluripotent stem (iPS) cells. Moreover, activation of combinatorial transcription factors also induces the generation of many cell types, such as cardiomyocytes, neurons, and hepatocytes, directly from somatic cells. These works indicate that single TFs are not sufficient to achieve a cell fate conversion process in most cases. Thus, a platform that allows combinatorial screen is in urgent need to facilitate cell fate determination studies.
To perform a second-round combinatorial activation screen, an sgRNA library that achieves double gene activation is generated. In this library, two different sgRNA cassettes are constructed into one vector. The first cassette contains sgRNAs targeting top hit genes from the single activation screen, which are driven by a human U6 promoter. Meanwhile, each vector contains the second cassette, which is a sgRNA with a different stemloop sequence driven by a mouse U6 promoter. The sgRNAs of the second cassettes also target top hit genes from the first round activation single screen. This construct expresses sgRNAs targeting two different genes, as well as avoids recombination of repeated sgRNA sequences. Two different sgRNAs bind to dCas9 and achieve the activation of two different top hit genes simultaneously in the dCas9-activation system. This allows the combinatorial double activation screen.
In some embodiments, this double activation library is transduced into CamES cells, and neural differentiation is performed as in the single screen. On day 12, cells are harvested and sorted for hCD8+ and hCD8−. The sgRNAs are sequenced and paired-analyze enriched genes in hCD8+ and hCD8− populations are identified. The screen identifies optimal TF combinations that drive neural differentiation of mouse ES cells.
Additionally, the combination of gain-of-function and loss-of-function techniques accelerates cell fate conversion, and sheds light on the fully revelation of cellular reprogramming mechanisms. However, a platform to perform gain-of-function and loss-of-function screen simultaneously is not available at present.
To perform a simultaneous activation/repression screen, a clonal ES cell line carrying gene activation/repression cassettes is generated. Vectors containing two cassettes separately are constructed. One vector contains the activation cassette, which is a dead Streptococcus pyogenes Cas9 (SpCas9)-activation system, with a eGFP gene cassette. The other vector comprises SadCas9-KRAB, with a zeocin-resistance gene cassette following. The two vectors, together with Cas9 and sgRNA targeting mouse Rosa26 loci are co-transfected into mouse ES cells. To select mouse ES cells carrying these two system, transfected ES cells are selected with zeocin. After seven days, remaining zeocin-resistant cells are analyzed with flow cytometry and single GFP+ cells are sorted into 96-well plates. One week later, the genome of clonal cells is analyzed to confirm the correct integration of both activation and repression cassettes. This clonal cell line allows the activation and repression of different genes simultaneously.
An sgRNA library that achieves gene turning-on and -off simultaneously is constructed. In this library, two different sgRNA cassettes are constructed into one vector. The first cassette contains sgRNAs of SpCas9 targeting top hit genes from the single activation screen, which are driven by a human U6 promoter. Meanwhile, each vector contains the second cassette, which is a sgRNA of SaCas9 driven by a mouse U6 promoter. The sgRNAs of SaCas9 in the second cassettes target top hit genes from the first round repression screen. This construct expresses sgRNAs of SpCas9 and SaCa9, and thus allows simultaneous gene activation and repression.
This activation/repression library is applied to clonal turning-on/off mouse ES cells, and neural differentiation is performed as in the single screen. On day 12, cells are harvested and sorted for hCD8+ and hCD8−. The sgRNAs and paired-analyze enriched genes in hCD8+ and hCD8− populations are sequenced. A series of gene combinations having both TF determinants and neural differentiation barriers is identified. The simultaneous turning-on of IT determinants and turning-off of neural differentiation barriers generates very high efficiency of neural cells of mouse ES cells.
Example 4 Experimental Procedures Plasmid Design and Construction To clone sgRNA vectors, the optimized sgRNA expression vector (pSLQ1373) was linearized and gel purified (Chen et al., 2013). New sgRNA sequences were PCR amplified from pSLQ1373 using different forward primers and a common reverse primer, gel purified and ligated to the linearized pSLQ1373 vector using In-Fusion cloning (Clontech). Primers used to construct individual sgRNAs are shown in Table 8. To change the promoter of scFv-sfGFP-VP64, the EF1α and PGK promoters were PCR amplified, gel purified, and ligated to linearized pSLQ504 using In-Fusion cloning (Clontech).
Two-guide expression vectors were assembled by a two-step cloning procedure. First, new sgRNA sequence (integrated DNA Technologieds) were PCR amplified from pSLQ5004 and ligated into BstXI and XhoI-digested pSLQ5004 parental vector, which contained a modified human 136 promoter (hU6). The same single sgRNA expression constructs were cloned into pSLQ1373 as previously described, which contained a modified mouse U6 promoter (mU6) and an optimized stem loop sequence of sgRNA. Second, the two-guide expression cassettes were then assembled from PCR amplified single cassettes using two sgRNA forward and reverse primers from pSLQ5004-based single sgRNA constructs and inserted into NsiI-digested pSLQ1373 single sgRNA constructs. Primers used to construct individual sgRNAs are shown in Table 11.
sgRNA Library Design
Putative transcription factor (TF) genes were selected according to the TRANSFAC database, and TSS (transcription start site) for each gene was determined using the Gencode and refFlat databases. All possible transcripts were selected if multiple TSSs exist for a gene. All sgRNAs targeting −3 kb to 0 relative to TSS were kept. Using the CRISPR-era algorithm (Liu et al., 2015), the targeting sequences of sgRNAs adjacent to an NGG PAM (protospacer adjacent motif) were computed, starting with a G (for more efficient U6 promoter activity) with a length of 20 bp. The sgRNAs containing homopolymers spanning greater than 3 nucleotides (nt) were discarded. To avoid off-target effects, sgRNA sequences alignment to the mouse genome was computed using the short read aligner Bowtie, and those with less than 2 mismatches with another genomic region were excluded. Furthermore, sgRNA sequences that contained certain restriction sites (BstXI and BlpI) were also removed. sgRNAs with a GC content between 30% and 70% were kept. An average of about 60 sgRNAs were selected for each target gene. Sequences for non-targeting negative control sgRNAs were generated using a randomized mouse gene TSS region and selected using the same rules as described above.
sgRNA Library Construction
The oligonucleotide pool was synthesized by Custom Array. The oligo library was PCR amplified, gel purified and ligated to the linearized backbone vector (pSLQ1373) digested with BstXI and BlpI using In-Fusion cloning. Libraries and parental vector will be made available on addgene.org.
Cell Culture E14 mouse ES cells and CamES cells were maintained on gelatin coated tissue culture plates with basal medium (50% Neurobasal, 50% Dulbecco modified Eagle medium (DMEM)/Ham's nutrient mixture F12, 0.5% NEAA, 0.5% Sodium Pyruvate, 0.5% GlutaMax, 0.5% N2, 1% B27, 0.1 mM β-mercaptoethanol and 0.05 g/L bovine albumin fraction V; all from Thermo Fisher Scientific) supplemented with LIF (Millipore) and 2i (Stemgent). Human embryonic kidney (HEK293T) cells (ATCC) were cultured in 10% fetal bovine serum (Thermo Fisher Scientific) in DMEM (Thermo Fisher Scientific).
Construction of the CamES Cell Line Mouse ES cells were co-transduced with multiple lentiviral constructs that expressed dCas9-SunTag from a TRE3G promoter, scFV-sfGFP-VP64 from the EF1a or PGK promoter, and reverse tetracycline-controlled transactivator (rtTA) from the EF1a promoter. After adding Doxycycline, polyclonal cells were sorted by flow cytometry using a BD FACS Aria2 for GFP+ and mCherry+ cells. After verification of gene activation using a sgBrn2, monoclonal cells were further sorted, and one efficient cell line was chosen as CamES cells.
Construction of the Tuj-1-hCD8 CamES Cell Line Construction of CRISPR/Cas9 vector for Tuj1 knockin. The pX330-derived pSLQ1654 encoding the nuclease Cas9 and an optimized sgRNA sequence was first linearized by a BbsI digest and gel purified. Two primers sgTuj-1 F and sgTuj-1 R were phosphorylated, annealed, and ligated to the linearized vector pSLQ1654 to generate pSLQ1654-sgTuj1. sgTuj-1 F: caccgcccaagtgaagttgctcgcagc. sgTuj-1 R: aaacgctgegagcaacttcacttgggc.
Construction of DNA template. The Tuj1-IRES-hCD8 vector (pSLQ1760) was assembled with three fragments (5′ homologous arm of Tuj1, IRES-hCD8 and 3′ homologous arm of Tuj1) and a modified pUC19 backbone vector by using Gibson Assembly Master Mix (New England Biolabs). Both 5′ and 3′ homology arms were PCR amplified from the genomic DNA extracted from mouse ES cells with Herculase 11 Fusion DNA polymerase (Agilent). The IRES-hCD8 was PCR amplified from pSLQ1729. The backbone vector was linearized by digestion with PmeI and ZraI. All DNA fragments and the backbone vector were gel purified followed by a Gibson assembly reaction. Primers: 5′ homologous arm F: aaagtgccacctgacactcagtcctagatgtcgtgegg (SEQ ID NO:380). 5′ homologous arm R: tcacttgggcccctgggct (SEQ ID NO:381). IRES-human CD8 F: caggggcccaagtgaactagtaaaattcgcccctctccctc (SEQ ID NO:382). IRES-human CD8 R: cagctgcgagcaactttaacctgcaaaaagggagcagtaaagg (SEQ ID NO:383). 3′ homologous arm F: agttgctcgcagctggggt (SEQ ID NO:384). 3′ homologous arm R: agctggagaccgttttttctgactgactggalacagggcat (SEQ ID NO:385).
Electroporation and clonal Tuj1-hCD8 CamES cells: 2.5 μg pSLQ1654-sgTuj1, 12.5 μg Tuj1-IRES-hCD8 template DNA in 100 μL Nucleofector solution (Amaxa) were electroporated into 1×106 CamES cells using program A-030. Both plasmids were maxiprepped using the Endofree Maxiprep Kit (Qiagen). After 3 days of culture, sorted single cells were seeded in a 96-well plate with one cell per well. All clonal cell lines were analyzed using PCR and sequencing (Yu et al., 2015).
Lentiviral Production HEK293T cells were seeded at ˜30% confluence one day before transfection. Lentivirus were produced by cotransfecting with pHR plasmids and encoding packaging protein vectors (pMD2.G and pCMV-dR8.91) using TransIT-LT1 transfection reagents (Mirus). Viral supernatants were collected 3 days after transfection and filtered through 0.45 μm strainer. Supernatant was used for transduction immediately or kept at −80° C. for long-term storage.
Quantitative RT-PCR Cells were harvested using Accutase (STEMCELL), and total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN), according to manufacturer's instructions. Reverse transcription was performed using iScript cDNA Synthesis kit (Bio-Rad). Quantitative PCR reactions were prepared with iTaq Universal SYBR Green Supermix (Bio-Rad). Reactions were run on a LightCycler thermal cycler (Bio-Rad). Primers used are summarized in Table 9.
High-Throughput Pooled Neural Differentiation Screens The neural differentiation screens were performed as two independent replicates. For both screens, 108 CamES cells were seeded at 40,000 cells/cm2 density at day −2. Cells were transduced with pooled lentiviral sgRNA library with an MOI of 0.3 at day −1 in basal medium supplemented with LIF and 2i. At day 0, puromycin was added at 1 μg/mL in ES2N medium (Millipore) with Doxycycline for another 24 hours. Fresh ES2N medium was changed with Doxycycline every day starting day 2. On day 12, cells were harvested and sorted for hCD8+ and hCD8− cells using EasySep human CD8 isolation kit (STEMCELL Technologies). Populations of cells expressing this library of sgRNAs were either harvested at the outset of the experiment (the day 0 time point: after 24 hours puromycin selection), hCD8+, or hCD8− cells. Genomic DNA was harvested from all samples; the sgRNA-encoding regions were then amplified by PCR using HiSeq forward and reverse primers and sequenced on an lllumina HiSeq-4000 using HiSeq custom primer with previously described protocols at high coverage (Bassik et al., 2013; Kampmann et al., 2014). Primers used are summarized in Table 12.
For the individual sgRNA validation experiments, a similar protocol was used, except that CamES cells were cultured in basal medium seeded at 5,500 cells/cm7 after puromycin selection and transduced with a high MOI. Top 100 hits are summarized in Table 10.
Combinatorial sgRNA Library Construction
A library of 44 sgRNAs including a set of 19 genes was designed by using the top prediction hits from the single screens and six nontargeting negative-control sgRNAs. Any sgRNAs containing NsiI restriction sites, which were used for combinatorial sgRNA library construction, were excluded. Individual oligonuclotides encoding sgRNAs were synthesized in a 96-well format (Integrated DNA Technologieds), and cloned into pSLQ1373 individually as previously described. At the same time, the same sgRNA sequence was synthesized (Integrated DNA Technologies) using different forward sequence. These sgRNAs were cloned into pSLQ5004 individually as previously described. After sequencing validation, all pSLQ1373-sgRNA constructs were manually mixed and all pSLQ5004-sgRNA constructs separately mixed in equal amounts for combinatorial sgRNA library construction. To generate the pooled combinatorial sgRNA library, the sgRNA sequence were PCR amplified using two sgRNA forward and reverse primers from pooled pSLQ5004-sgRNA constructs, gel purified and ligated into the NsiO-digested pooled pSLQ1373-sgRNA constructs using In-Fusion cloning (Clontech). The combinatorial sgRNA-library pools were prepared in Stellar competent cells (TaKaRa) and purified with a Plasmid Maxi Kit (Qiagen). The representation of each of the double-sgRNA constructs was then quantified by NGS with the oligonucleotides listed in Table 11.
High-Throughput Pooled Combinatorial Screens The double neural differentiation screens were performed as two independent replicates. For both screens, 6 millions CamES cells were seeded at 40,000 cells/cm2 density at day −1. Cells were transduced with pooled lentiviral double sgRNA library with an MOI of 0.3 at day 0 in basal medium supplemented with LIF and 2i. At day 1, puromycin was added at 1 μg/mL in basal medium with Doxycycline for another 24 hours. Fresh basal medium was changed with Doxycycline every day starting day 2. On day 12, cells were harvested and sorted for CD8+ and CD8− cells using Aria II cell sorter (BD Biosciences). Genomic DNA was harvested from all samples; the double sgRNA-encoding regions were then amplified by PCR using MiSeq forward and reverse primers and sequenced on an Illumina Miseq using HiSeq custom primer, which for the first sgRNA, and MiSeq custom primer, which for the second sgRNA. Primers used are summarized in Table 12.
For the individual double sgRNA validation experiments, a similar protocol was used, except that CamES cells were transduced with a high MOI.
Primary Neurons Culture, Primary Astrocytes Culture and Induced Neurons Replating Primary cultures of cortex neurons were prepared from postnatal day 1 wild-type black rat. Rats were decapitated, and their brains were removed in pre-cooled physiological saline. The cortex was dissected. Tissues were slightly minced and placed into a Papain Dissociation solution (Worthington Biochemical Corporation) containing 20 units/ml papain and 0.005% DNase in Earle's Balanced Salt Solution (Thermo Fisher Scientific). The solution was equilibrated in 95% O2, 5% CO2 before the tissue was incubated at 37° C. for 1 hour. After incubation, the tissue and solution mixture was triturated. Undissociated tissue was allowed to settle and the cloudy suspension was removed and centrifuged at 300×g for 5 minutes. The supernatant was then discarded and the cell pellet was resuspended in a DNase/albumin-inhibitor solution. A discontinuous density gradient was prepared by gently layering the cell suspension on top of an albumin-inhibitor solution in a centrifuged tube. The mixture was centrifuged at 145×g for 5 minutes. The supernatant was discarded and the neurons were resuspended in Neurobasal (Invitrogen) medium containing 2% B27 supplement, 2 mM glutamine and 0.5% penicillin/streptomycin. A total of 250,000 cells were plated onto a well of 24-well plates that had been pre-treated with 12.5 μg/ml poly-D-lysine (Sigma). The plates were incubated at 37° C. in a 5% CO2/95% air incubator and half of the medium was changed every three days.
Rat Primary Cortical Astrocytes (Thermo Fisher Scientific) were cultured and plated according to manufacturer's instructions. The astrocytes were fed every three days with fresh medium.
One week after culturing primary neurons and astrocytes, the induced neurons were gently removed from the dishes by trypsin dissociation and were replated onto primary neurons or astrocytes. Electrophysiological recordings were performed between day 14 and day 21 after replating.
Generation of Induced Neurons Preparation Before Induction
-
- 1. Embryonic skin-derived fibroblasts were isolated from E13.5 embryos of C57BL/6 mice as previously described (2010 nature, Vierbuchen et al.). Isolated fibroblasts were cultured and expanded in MEF media (Dulbecco's Modified Eagle Medium, Life Technologies) containing 10% Fetal Bovine Serum (Life Technologies), non-essential amino acids (Life Technologies), and sodium pyruvate (Life Technologies)) for 2 passages before use. Tail tip fibroblasts were isolated from the bottom third of tails from 4-day-old pups as previously described. Tail tip cells were expanded for 2 passages in MEF media before use.
- 2. Matrigel (growth factors reduced; BD Biosciences) was thawed on ice according to the manufacturer's instruction and dilute it in pre-cold PBS with a ratio of 1:30.
- 3. Diluted matrigel was added to 24-well plates. It was ensured that the quantity used was sufficient to cover the entire growth surface of the plates and keep the plates in 37° C. for 30 minutes to be ready to use.
- 4. Passage 1-2 MEFs were thawed and seeded into the matrigel-coated plates at a preferentially density of 25,000 cells per well of a 24-well plates. Cells were grown in the MEF medium for 4-5 days until confluent.
Induction of Induced Neurons
-
- 1. When MEFs were grown confluent, cells were infected with lentiviruses containing expression constructs of rtTA (driven by ubiquitin promoter) and additional lentiviruses overexpressing Asc11-Neurog1/Ezh2-Foxo1/Brn2/Nr4a1/Dmrt3/Jun/Suz12/Nr3c1/Tcf15/Zeb1/Mecom/Hoxc 8/Nr2f1 (driven by Tet-on promoter) in the presence of polybrene (8 mg/ml).
- 2. The next day, media was exchanged with basal medium (50% Neurobasal, 50% Dulbecco modified Eagle medium (DMEM)/Ham's nutrient mixture F12, 0.5% NEAA, 0.5% Sodium Pyruvate, 0.5% GlutaMax, 0.5% N2. 1% B27, 0.1 mM β-mercaptoethanol and 0.05 g/L bovine albumin fraction V; all from Thermo Fisher Scientific) containing doxycycline (2 mg/ml).
- 3. Culture medium was refreshed every 3-4 days during the induction period.
Maturation of Induced Neurons After lentiviruses infection for about 14 days (extensive neurites outgrowth should be observed in this stage), the induced cells were progressed for further maturation: Re-plate and co-culture directly with primary neurons/astrocytes.
-
- 1. Mouse primary postnatal cortical neurons or astrocytes were isolated and cultured for about 6 days before re-plating the induced cells.
- 2. The induced cells were dissociated by using 0.05% trypsin from the culture plate.
- 3. Cells were centrifuged for 3 min at 1000 rpm at room temperature.
- 4. The supernatant was discarded, fresh differentiation medium (basal media with addition of 200 μM ascorbic acid, 2 μM db-cAMP, 25 ng/ml BDNF, 25 ng/ml NT3, and 50 ng/ml GDNF) was added to gently re-suspend the cells and cells were re-plated to co-culture with pre-existing primary neurons/primary astrocytes.
- 5. Re-plated cells were co-cultured for about 14 days or longer (depending on the maturation process of the induced cells, which can be observed based on the development of the extensive neuritis outgrowth) to become functional mature. Half of the maturation medium was changed every 2-3 days.
Flow Cytometry, Cell Surface Staining and Cell Sorting The antibody CD8-APC was purchased from BD Biosciences. and Anti-PSA-NCAM-APC was from Miltenyi Biotec. Cells were harvested, washed, and adjusted to a concentration of 106 cells/mL in ice cold PBS with 2% FBS. Cells were stained and incubated with diluted primary antibodies at 4° C. for 30 mins in Eppendorf tubes. After staining, cells were washed three times by centrifugation at 400 g for 5 mins and resuspended in 500 μL to 1 mL in ice cold PBS. Cells were kept in dark on ice and analyzed using BD Accuri C6 Cytometer. Cell sorting was performed by using Aria II cell sorter (BD Biosciences).
Immunocytochemistry Experiments were performed on cells seeded on plate (IBIDI) that had been coated with gelatin (0.1%) overnight at 37° C. Cells were washed twice with PBS, fixed in 4% Paraformaldehyde (Wako) for 15 mins at room temperature, permeabilized and blocked with 0.1% Triton X-100, 5% donkey serum in PBS (blocking buffer) for 1 h at room temperature. After three times wash with PBS, cells were incubated with primary antibodies. The following primary antibodies with indicated dilution in blocking buffer were used: Rabbit anti-Oct4 (Santa Cruz, 1:200), Mouse anti-Tuj1 (Covance, 1:1000), Rabbit anti-Map2 (Cell Signaling Technology, 1:200), Rabbit anti-NeuN (Abcam, 1:1000), Rabbit anti-vGluT1 (Synaptic Systems, 1:200), Rabbit anti-GFAP (Dako, 1:500), Rabbit anti-Olig-2 (Millipore, 1:500), Rabbit anti-Tbr1 (Abcam, 1:100), Rabbit anti-Synapsin I (Abcam, 1:200), Rabbit anti-GABA (Sigma, 1:250). Cells were incubated with primary antibodies at 4° C. for overnight, then washed three times with PBS. After staining with corresponding secondary antibodies in blocking buffer for 1 hour at room temperature, cells were washed three times with PBS and stained with DAPI (Vector Labs) for 5 mins. Washed cells were examined using a Nikon Spinning Disk Confocal microscope with TIRF.
Efficiency Calculation The following method was used to calculate the efficiency of neuronal induction. The total number of Map2+ cells with a neuronal morphology, defined as cells having a circular, three-dimensional appearance that extend a thin process at least three times longer than their cell body, were quantified 14 days after infection. The Map2+ and DAPI+ cells were counted from at least 20 randomly selected images at 20× magnification for each condition. The Map2+ cell number was divided by the number of DAPI+ cells to get a percentage of neuron-like cells.
Electrophysiology Lentivirus infections (with an additional sfGFP-expression virus) and transgene induction were performed similarly to as described for the fibroblast-induced neurons production, using basal medium. Patch-clamp electrophysiological recordings were performed on sfGFP positive fibroblast-induced neurons. GFP positive neurons located using a Lambda DG-4 illumination system and Q Imaging Fast 1394 Rolera-Mgi Plus camera controlled by Micro-Manager (Version 1.4) mounted on an Olympus BX51WI fluorescence microscope. Whole-cell responses were recorded using an MultiClamp 7008 (Molecular Devices) amplifier and headstage and low-pass filtered at 10 KHz before digitization using a DigiData 1440 data acquisition system (Molecular Devices). Data was stored on a PC running pClamp software (Version 10.4, Molecular Devices). Patch-pipettes were fabricated from 1.5 mm OD borosilicate capillary glass (Warner Instruments) using a microipette puller (Sutter Instrument, Model P-87) to give tip resistances of 2-4 MO. The series resistance for all recordings was under 10MΩ (Mean: 5.62MΩ, SEM: 0.38, n=12). Capacitance transients and series resistance errors were compensated for (70%) using the amplifier circuitry. The sodium and potassium currents currents were recorded in the voltage-clamp configuration at a holding potential of −80 mV. Spontaneous postsynaptic currents were recorded in the voltage-clamp configuration at a holding potential of −60 mV or −70 mV. Spontaneous action potentials were recorded in neurons held at −60 mV to −80 mV. Action potentials were also evoked by applying depolarizing current.
All experiments were performed at ambient room temperature (25° C.). The external solution contained (in mM): NaCl (130), HEPES-Na (10), KCl (5), CaCl2(2), Glucose (10). For voltage-gated sodium currents, tetraethylammonium (TEA, 5 mM) was added to the external solution and the internal solution contained (in mM): CsF (120), HEPES (10), EGTA (11), CaCl2 (1), MgCl2 (1), TEA-Cl (10), KOH (11). For voltage-gated potassium currents, tetrodotoxin (TTX, 500 nM) was added to the external solution and the internal solution contained (in mM): KF (120), HEPES (10), EGTA (11), CaCl2) (1), MgCl2 (1), KCl (10), KOH (11). For current clamp recordings of action potentials, 2 mM MgATP was added to the internal solution. All recording solutions had pH values of 7.3-7.4 with osmolality of 290-300 mOsm/kg. Drug applications were administered via local perfusion approximately 200 μm from the recorded cells at a flow rate of 0.2 ml/min and solutions were continually withdrawn from the recording chamber by vacuum aspiration. Drugs were applied until responses reached a steady-state level. Electrophysiological data were analyzed offline using Clampfit 10.4 and data was plotted using Graphpad Prism software.
Bloinformatic Analysis of sgRNA and Gene Hits
Data processing was conducted with custom scripts. Reads were mapped allowing for a mismatch for the first and last base pair of the spacer, which uniquely identified sgRNA. Each sample was normalized by the total read count. This gave a frequency for each sgRNA:
The paired Tuj1-hCD8+ and Tuj1-hCD8− were used to compute the enrichment scores. Specifically, frequencies as above were computed as above, and sgRNA with less than 1 count in the Tuj1-hCD8− library were discarded. Enrichment was computed for each sgRNA in each replicate as the log 2 fold-change from the Tuj1-hCD8− sample to the Tuj1-hCD8+ libraries. Enrichment was averaged across replicates and used as Esg in subsequent analysis. For each gene, an enrichment score (ESgene) was computed from the sgRNA enrichment above, as follows. An unnormalized enrichment score (Egene.top3) was computed by averaging Esg for the 3 sgRNA with highest Esg. Egene.top3 was normalized by the distribution of nontargeting sgRNA as follows (Gilbert et al., 2014, supra).
Suppose a gene had N targeting sgRNA. 10000 bootstrap samples of size N were drawn from the nontargeting sgRNA. For each sample of size N, Esample.top3 was computed as above. This gave an empirical estimate of the distribution of Egene.top3 if the all the sgRNA targeting that gene had been negative control sgRNA. For the final, normalized gene enrichment score (ESgene), the unnormalized enrichment score was divided by the 0.9 quantile of this empirical distribution:
After ranking genes by ES, the most enriched sgRNA was selected for each gene to subsequently validate.
Bioinformatic Analysis of Double Screen The count matrix was calculated by exact match for both ends, throwing all other reads out. The correlation of counts between replicates of the same condition was high (0.942-0.992), indicating high reproducibility of the double screen. Effect sizes for each gene pair was calculated using MAGeCK MLE (Li et al Genome Biology 2015, 16:281).
Suppose the null hypothesis that the guide pair of genes i and j have an effect size equal to the maximum of the individual effect size. This will be the case if one gene is the primary driver of neuronal differentiation. Note that the coefficients estimated by MACeCK (βij for genes i and j, in that order) arise from a generalized linear regression and should, if the model posited by MACeCK is correct, be normally distributed.
Consider the null hypothesis H0: the effect of guide targeting two genes is less than the maximal effect of guides targeting either gene. The order of the guide is taken into account. A consistent but smaller effect is predicted with the order of the guides reversed. Let signm(x, y) be the function that returns the sign of the larger of the absolute values of the inputs. Under the null hypothesis βij=signm(βi0, β0j) max(|βi0|, |β0j|).
To this end, note that the standard deviation of βij is bounded above by
√{square root over (8βi02+8β0j2)}.
Therefore the difference βij−signm(βi0, β0j) max(|βi0|, |β0j|) has standard error bounded above by
One can construct a test statistic to test H0 as
The test statistic constructed does not have an exactly normal distribution due to the high correlation between estimates (since all gene-gene pairs are tested) and therefore an empirical Bayes approach is used to determine significant genes while appropriately controlling the false discovery rate (Efron Large-scale inference: empirical Bayes methods for estimation, testing, and prediction, volume 1. Cambridge University Press. 2012; Efron et al R package 2011).
Determinants of CRiSPRa Guide Activity Since large variation gene effect size was observed (FIG. 27C) and an apparent mixture distribution in the top hits, a Bayesian hierarchical logistic regression mixture model was fit using stan (Carpenter et al 2017 J. of Stat. Software, Volume 76, Issue 1). Specifically, the following model was fit.
-
- xi=log2 fold change of guide i;
- gi=gene associated with guide i;
- xi˜ZiN(μgi, σ2)+(1−Zi)N(0, 1.42);
- μg˜ N(3, 1.52);
- Zi˜ Bernoulli(qi);
- yij=Indicator variable if guide i is in feature j;
- gci=GC content of guide i;
- di=distance from the TSS for guide i;
-
- βj˜Laplace(0.2);
- β0˜N(0, 5).
In this mixture model, features have a linear effect on the log-odds that the guides belong to the second component. In this way one can separate out gene-specific effects and compare guides targeting the same genes, but pooling the information across all genes. To shrink the feature effects towards zero, a Laplace prior is used. Eight chains were fit and good mixing in all chains and Rhat values near 1 was observed, indicating a good fit of the model.
TABLE 8
Primers sgRNA sequence
pSLQ1373- gtatcccttggagaaccaccttgttgnnnnnnn
Forward nnnnnnnnnnnnngttaagagctaagctggaaa
primer cagca (SED ID NO: 386)
pSLQ1373- gatcctagtactcgagaaaaaaagcaccgactc
Reverse ggtgccac
primer (SEQ ID NO: 387)
sgAscl1 gaatggagagtttgcaaggag
(SEQ ID NO: 401)
sgNeurog1-1 ggctgctgggagttgtgcaa
(SEQ ID NO: 405)
sgNeurog1-2 gtgcactactgaatccaaga
(SEQ ID NO: 530)
sgNeurog1-3 gtcaatcagtagcaggcaaa
(SEQ ID NO: 531)
sgMyod1 ggtctccagagtggagtccg
(SEQ ID NO: 406)
sgFoxo1-1 ggttcaggatgagtggaggc
(SEQ ID NO: 425)
sgFoxo1-2 gaagacttcactcatcttgg
(SEQ ID NO: 532)
sgFoxo1-3 gtctcagcgatcggattgct
(SEQ ID NO: 533)
sgNr2f1-1 ggagccaagagaagggctgc
(SEQ ID NO: 426)
sgNr2f1-2 gaagtatatcatagtttcgg
(SEQ ID NO: 534)
sgNr2f1-3 gtttggagtttgagcatcct
(SEQ ID NO: 535)
sgBrn2-1 gaggaaggactgagaagact
(SEQ ID NO: 428)
sgBrn2-2 gtgtaagggatctttgttac
(SEQ ID NO: 536)
sgBrn2-3 gtgtttatgaaagtgtatgg
(SEQ ID NO: 537)
sgEzh2-1 ggttcctttcggcaccttgg
(SEQ ID NO: 429)
sgEzh2-2 gataactgaacagggagtgg
(SEQ ID NO: 538)
sgEzh2-3 gttcggccctctgattggac
(SEQ ID NO: 539)
sgNr4a1-1 gctaacgtgtagtctcgttg
(SEQ ID NO: 431)
sgNr4a1-2 gccacctaggagaagaagtg
(SEQ ID NO: 540)
sgNr4a1-3 ggtttcctttagcttagact
(SEQ ID NO: 541)
sgDmrt3-1 gaggagttgatagttgttcc
(SEQ ID NO: 433)
sgDmrt3-2 gttacaatagactttgaggc
(SEQ ID NO: 542)
sgDmrt3-3 ggcaggtattaatactcaag
(SEQ ID NO: 543)
sgJun-1 gagaataaagtgttgtgccg
(SEQ ID NO: 435)
sgJun-2 gtttacatccaggctttgag
(SEQ ID NO: 544)
sgJun-3 gtttggctgtctagtgacgg
(SEQ ID NO: 545)
sgSuz12-1 gaagctctcaaggcgagaaa
(SEQ ID NO: 436)
sgSuz12-2 gattctgtggaattgggttg
(SEQ ID NO: 546)
sgSuz12-3 gctcagtctcatctccactg
(SEQ ID NO: 547)
sgNr3c1-1 gtcactgctctttaccaaga
(SEQ ID NO: 438)
sgNr3c1-2 gttatggtttcaggctggaa
(SEQ ID NO: 548)
sgNr3c2-3 gactcttctgctcagtttgc
(SEQ ID NO: 549)
sgTcf15-1 gggatatgctcactttggga
(SEQ ID NO: 439)
sgTcf15-2 ggtcgtcgccttatagccgg
(SEQ ID NO: 550)
sgTcf15-3 gaagtgacaggatcagctat
(SEQ ID NO: 551)
sgZeb1-1 gaaggaactaagtttcttct
(SEQ ID NO: 440)
sgZeb1-2 gtgacaggtgatctaggcgc
(SEQ ID NO: 552)
sgZeb1-3 ggaaccttgttgctagggcc
(SEQ ID NO: 553)
sgMecom-1 gattctcaggcagggctcta
(SEQ ID NO: 442)
sgMecom-2 gaccagttcactgaaagatg
(SEQ ID NO: 554)
sgMecom-3 ggcagttctcttgcctagtg
(SEQ ID NO: 555)
sgHoxc8-1 gctctttcctctaacagccc
(SEQ ID NO: 443)
sgHoxc8-2 gaggtgagagttagtaagtc
(SEQ ID NO: 556)
sgHoxc8-3 gtcatcaaagaaagaatggc
(SEQ ID NO: 557)
TABLE 9
SEQ
Gene ID
name Primer sequence NO:
RiboL7 F accgcactgagattcggatg 444
RiboL7 R gaaccttacgaacctttgggc 445
Ascl1 F aagaagatgagcaaggtggagacg 446
Ascl1 R gagatggtgggcgacagga 447
Brn2 F tttcctcaaatgccctaagc 448
Brn2 R ggaggggtcatccttttctc 449
Tuj1 F agtcagcatgagggagatcg 450
Tuj1 R agtcccctacatagttgccg 451
Map2 F agcactgattgggaagcact 452
Map2 R caattcaaggaagttgtaaagtagt 453
gaagtttg
Foxo1 F gagtggatggtgaagagcgt 490
Foxo1 R tgctgtgaagggacagattg 491
Nr2f1 F ccaacaggaactgtcccatc 492
Nr2f1 R attcttcctcgctgaaccg 493
Neurog1 F cggcttcagaagacttcacc 494
Neurog1 R ggcctagtggtatgggatga 495
Pou3f2 F tttcctcaaatgccctaagc 498
Pou3f2 R ggaggggtcatccttttctc 499
Ezh2 F acttctgtgagctcattgcg 500
Ezh2 R cgactgcattcagggtcttt 501
Nr4a1 F gctagaaggactgcggagc 504
Nr4a1 R attgagcttgaatacagggca 505
Dmrt3 F agcgcagcttgctaaacc 508
Dmrt3 R gcttttgacaacatctgggg 509
Jun F gaaaagtagcccccaacctc 512
Jun R aatcagacaggggacacagc 513
Suz12 F tcgaaattccagaacaagca 514
Suz12 R tgtggaagaaaccggtaaatg 515
Nr3c1 F ggacaacctgacttccttgg 518
Nr3c1 R ctggacggaggagaactcac 519
Tcf15 F tctgcaccttctgtctcagc 520
Tcf15 R aaccagggatccaggttcat 521
Zeb1 F acagagaatggaatgtatgcatgtg 522
Zeb1 R agattccacactcgtgaggc 523
Mecom F acagcatgagatccaaaggc 526
Mecom R ttatcccatctgcatcagca 527
Hoxc8 F aaatcctccgccaacactaa 528
Hoxc8 R tgtaagtttgtcgaccgctg 529
TABLE 10
Enrichment
Rank Gene name score
1 Foxo1 2.49122811
2 Nr2f1 2.448600182
3 Neurog1 2.43849068
4 Rb1 2.435300527
5 Pou3f2 2.385360453
6 Ezh2 2.380072461
7 Maz 2.361103604
8 Nr4a1 2.351837703
9 Arnt 2.317336958
10 Dmrt3 2.304207908
11 Sin3b 2.280599668
12 Jun 2.277732884
13 Suz12 2.276236754
14 Klf12 2.269476929
15 Nr3c1 2.249983644
16 Tcf15 2.229200027
17 Zeb1 2.221200461
18 Nr6a1 2.208496165
19 Mecom 2.207944981
20 Trim24 2.206262504
21 Hoxc8 2.184103377
22 Foxk1 2.171388615
23 2410080102Rik 2.171161939
24 Nr4a3 2.168779599
25 Trp73 2.16579857
26 Foxs1 2.162897697
27 Ikzf3 2.15938851
28 Nkx2-6 2.15063949
29 Sox11 2.140964961
30 1110054M08.Rik 2.139005342
31 Crem 2.133968618
32 Meis3 2.131453549
33 Bmyc 2.130409666
34 Epas1 2.129339686
35 Nr2f6 2.128397081
36 Nacc1 2.120269011
37 Bsx 2.120136772
38 Foxd3 2.114601186
39 Myog 2.107435864
40 Smad3 2.105254748
41 Wt1 2.091731056
42 Taz 2.091306567
43 Smad7 2.071136269
44 Stra13 2.06971649
45 Hoxc4 2.062634453
46 Pou3f3 2.058607569
47 Zbtb12 2.051837502
48 Atf5 2.042025795
49 Gtf2a2 2.041587014
50 Pura 2.040735147
51 Snai1 2.040229657
52 Ncor1 2.038396405
53 Pcbp2 2.036271048
54 E2f2 2.028758908
55 Nfkbib 2.023153101
56 Gli2 2.021010016
57 Nr0b1 2.020715359
58 B230110C06Rik 2.016733057
59 T 2.014396786
60 Runx3 2.011724145
61 Rxra 2.011600497
62 Mafk 2.009964981
63 Foxn1 2.006315586
64 Smad4 1.999197443
65 Meis2 1.998728368
66 Hoxa1 1.996287157
67 Zic1 1.992579239
68 Sebox 1.99248237
69 Nfyc 1.983084664
70 Lmx1b 1.980716237
71 Lhx3 1.979175342
72 Hmx2 1.978886945
73 Arf6 1.977331424
74 Nfatc3 1.975872129
75 Neurod6 1.973516686
76 Smarca4 1.972359038
77 Twist1 1.971479015
78 Gzf1 1.963483117
79 Hoxc10 1.962998475
80 Tbx4 1.962626034
81 Npas2 1.962608209
82 Ctbp1 1.960624385
83 Gem2 1.960206991
84 Is12 1.957324105
85 Arid5a 1.956887379
86 Lef1 1.955552772
87 RP24-399L6.2 1.953337042
88 Smad5 1.949029539
89 Lbx1 1.948838891
90 Pax3 1.945680745
91 Foxj1 1.944149198
92 Tbx5 1.943975816
93 Barh11 1.943598679
94 Hoxd11 1.9410811
95 Poulf1 1.939557398
96 Klf3 1.938997548
97 Pebp1 1.937292841
98 Evx2 1.935442174
99 Irx5 1.934100096
100 Nkx6-3 1.928635054
TABLE 11
pSLQ5004- tggaaagccagaaacatgnnnnnnnnnnnnnnn
Forward nnnnngttttagagctagaaatagcaagttaaa
primer ataaggctagtcc
(SEQ ID NO: 558)
pSLQ5004- gatcctagtactcgaggtacctctaggc
Reverse (SEQ ID NO: 559)
primer
Two accgtattaccgccagccttttgctcattaat
sgRNA taaggtaccgagg
forward (SEQ ID NO: 560)
primer
Two TGACGGGCACatgcatggtacctctaggctag
sgRNA cgaattcAAAAAAAg
reverse (SEQ ID NO: 561)
primer
Scramble GAACGACTAGTTAGGCGTGTA
sgRNA-1 (SEQ ID NO: 562)
Scramble GTTTAGTAGTTCGTCACACC
sgRNA-2 (SEQ ID NO: 563)
Scramble GCGACATGTCTGTTGGGCGA
sgRNA-3 (SEQ ID NO: 564)
Scramble GTATATAAGCCGGGCGCACG
sgRNA-4 (SEQ ID NO: 565)
Scramble GTCGAACCACGCGTTGATCG
sgRNA-5 (SEQ ID NO: 566)
Scramble GACCCATGACGGTCGACGGA
sgRNA-6 (SEQ ID NO: 567)
sgFoxo1-1 GGTTCAGGATGAGTGGAGGC
(SEQ ID NO: 568)
sgFoxo1-2 GAAGACTTCACTCATCTTGG
(SEQ ID NO: 532)
sgNr2f1-1 GGAGCCAAGAGAAGGGCTGC
(SEQ ID NO: 426)
sgNr2f1-2 GAAGTATATCATAGTTTCGG
(SEQ ID NO: 534)
sgNeurog1-1 GTGCACTACTGAATCCAAGA
(SEQ ID NO: 405)
sgNeurog1-2 GTGCACTACTGAATCCAAGA
(SEQ ID NO: 530)
sgRb1-1 GGCTACATACAGTCTAGGTT
(SEQ ID NO: 427)
sgRb1-2 GAGGAATCGAGAACTTAATT
(SEQ ID NO: 569)
sgPou3f2-1 GAGGAAGGACTGAGAAGACT
(SEQ ID NO: 428)
sgPou3f2-2 GTGTAAGGGATCTTTGTTAC
(SEQ ID NO: 536)
sgEzh2-1 GGTTCCTTTCGGCACCTTGG
(SEQ ID NO: 429)
sgEzh2-2 GATAACTGAACAGGGAGTGG
(SEQ ID NO: 538)
sgMaz-1 GGAAGGCATCTCTGGGAAGC
(SEQ ID NO: 430)
sgMaz-2 GCTCTGCAGGACACCCATGT
(SEQ ID NO: 570)
sgNr4a1-1 GCTAACGTGTAGTCTCGTTG
(SEQ ID NO: 431)
sgNr4a1-2 GCCACCTAGGAGAAGAAGTG
(SEQ ID NO: 540)
sgDmrt3-1 GAGGAGTTGATAGTTGTTCC
(SEQ ID NO; 433)
sgDmrt3-2 GTTACAATAGACTTTGAGGC
(SEQ ID NO: 542)
sgSin3b-1 GTGCAAGAATTCAGTCCACA
(SEQ ID NO: 434)
sgSin3b-2 GTGGTCAAGGTACACACCTA
(SEQ ID NO: 571)
sgJun-1 GAGAATAAAGTGTTGTGCCG
(SEQ ID NO: 435)
sgJun-2 GTTTACATCCAGGCTTTGAG
(SEQ ID NO: 544)
sgSuz12-1 GAAGCTCTCAAGGCGAGAAA
(SEQ ID NO: 436)
sgSuz12-2 GATTCTGTGGAATTGGGTTG
(SEQ ID NO: 546)
sgKlf12-1 GATTTGACCATCTCTTGCCG
(SEQ ID NO: 437)
sgKlf12-2 GAGTCACATTGATCCTGCAA
(SEQ ID NO: 572)
sgNr3c1-1 GTCACTGCTCTTTACCAAGA
(SEQ ID NO: 438)
sgNr3c1-2 GTTATGGTTTCAGGCTGGAA
(SEQ ID NO: 548)
sgTcf15-1 GGGATATGCTCACTTTGGGA
(SEQ ID NO: 439)
sgTcf15-2 GGTCGTCGCCTTATAGCCGG
(SEQ ID NO: 550)
sgZeb1-1 GAAGGAACTAAGTTTCTTCT
(SEQ ID NO: 440)
sgZeb1-2 GTGACAGGTGATCTAGGCGC
(SEQ ID NO: 552)
sgNr6a1-1 GATGACGGTCGGCCGTAGTT
(SEQ ID NO: 441)
sgNr6a1-2 GAATCAGGAAGGCTGTAGCA
(SEQ ID NO: 573)
sgMecom-1 GATTCTCAGGCAGGGCTCTA
(SEQ ID NO: 442)
sgMecom-2 GACCAGTTCACTGAAAGATG
(SEQ ID NO: 554)
sgHoxc8-1 GCTCTTTCCTCTAACAGCCC
(SEQ ID NO: 443)
sgHoxc8-2 GAGGTGAGAGTTAGTAAGTC
(SEQ ID NO: 556)
sgOct4-1 GTCTGGACAGGACAACCCTT
(SEQ ID NO: 574)
sgOct4-2 GAGTGCCTGTCTGCAAGGGA
(SEQ ID NO: 575)
sgNanog-1 GGAAGTTTCAGGTCAAGTGG
(SEQ ID NO: 407)
sgNanog-2 GCTGTAAGGTGACCCAGACT
(SEQ ID NO: 576)
sgEsrrb-1 GGTTAGTGGGCTCCAAGTGT
(SEQ ID NO: 577)
sgEsrrb-2 GGTGAGTGAGTGACACCCTC
(SEQ ID NO: 578)
sgKlf2-1 GAAAGGACCTGTGGACAGTT
(SEQ ID NO: 579)
sgKlf2-2 GCAAGAGGGTAATAGAGAGA
(SEQ ID NO: 580)
TABLE 12
HiSeq aatgatacggcgaccaccgagatctacacagat
forward cggaagagcacacgtctgaactccagtcacnnn
primer nnngcacaaaaggaaactcaccct
(SEQ ID NO: 581)
HSeq caagcagaagacggcatacgagatcgactcggt
reverse gccactttttc
primer (SEQ ID NO: 582)
HiSeq gtgtgttttgagactataagtatcccttggaga
custom accaccttgttg
primer (SEQ ID NO: 583)
(the
first
sgRNA)
MiSeq aatgatacggcgaccaccgagatctacacagat
forward cggaagagcacacgtctgaactccagtcacnnn
primer nnngcacaaaaggaaactcaccct
(SEQ ID NO: 581)
MiSeq caagcagaagacggcatacgagatggtacctct
reverse aggctagcgaattc
primer (SEQ ID NO: 584)
MiSeq ccactttttcaagttgataacggactagcctta
custom ttttaacttgctatttctagctctaa
primer (SEQ ID NO: 585)
(the
second
sgRNA)
Results CRISPRa Screening Strategy for Neuronal-Fate-Inducers This example describes the identification of novel TFs driving direct neuronal reprogramming from fibroblasts. Using primary fibroblasts as a screening platform is technically challenging. Firstly, as primary cells have limited expansion capacities, it is difficult to modify them to generate a homogenous population, which achieves consistent CRISPR activation activities. Secondly, the neuronal transdifferentiation of fibroblasts is inefficient and not well suited for the enrichment of the desired cell population for the subsequent sgRNA sequencing.
Thus, mouse ES cells were chosen as a screening platform for the generation of candidate TFs driving neuronal-fate. The ectopic expression of individual key TFs that are critical for neuronal transdifferentiation can also drive neuronal differentiation of mouse ES cells, which supports the use of mouse ES cell differentiation as a discovery tool for neuronal-inducing TFs. Besides, as a model of developmental biology, ES cells have been successfully used to elucidate roles of many master transdifferentiation TFs of other lineages. Finally, mouse ES cells are technically easy to be equipped with CRISRP activation tools and suitable for single sgRNA screens.
A polypeptide-based SunTag CRISPRa system in mouse ES cells (Tanenbaum et al., 2014, supra) was modified (FIG. 22A). After several rounds of optimization and clonal cell selection based on endogenous gene activation efficiency, a CRISPR-activating mouse ES (CamES) cell line containing lentivirus-transduced CRISPRa elements was generated (FIG. 22B). Next, the CamES cell line was modified with a neuronal reporter. The reporter CamES cell line carrying a biallelic human CD8 (hCD8) gene cassette appended downstream to endogenous Tuj1 via an IRES (internal ribosome entry site) (Tuj1-hCD8 CamES) (FIGS. 22C and 22D). The magnetic-activated cell sorting (MACS)-enriched differentiated hCDS+ cells expressed much higher neuronal markers (Tuj1 and Map2) than hCD8− cells (FIG. 22E), demonstrating that hCD8 expression is positively correlated with differentiated neuronal cells.
An Unbiased Screen for Key Factors Promoting Neuronal Differentiation An sgRNA library targeting all putative TFs (˜800), with an average of 60 sgRNAs per gene was constructed. This sgRNA library also contained 9,296 non-targeting negative control sgRNAs, leading to a total of 55,336 sgRNAs (FIG. 18A). The sgRNA library was transduced into Tuj1-hCD8 CamES cells and 2i+Lif was removed from ES medium to allow neuronal differentiation (FIG. 23A). The Tuj1-hCD8 CamES cells showed highest neuronal marker expression between day 10 and 11 post-transduction (FIG. 23B). MACS were used to sort Tuj1-hCD8+ and Tuj1-hCD8− populations on day 12 (FIG. 23C), and the sgRNA distributions of these two samples were compared, as well as the plasmid library (FIG. 18A). The Tuj1-hCD8+ and Tuj1-hCD8− cell populations exhibited similar sgRNA depletion when compared to plasmid library (Figure S2D). A high correlation of enriched genes between the positive and negative Tuj1-hCD8 populations was found (FIG. 23E). The top hits relative to the plasmid pool in both populations contain many proliferation and self-renewal genes, but few are related to neuronal phenotypes (FIG. 23E). It was contemplated that this is because the predominant factors that determine sgRNA representation in both the Tuj1-hCD8+ and Tuj1-hCD8− populations are in common, such as the growth advantage of cells expressing proliferation and self-renewal genes, less proliferative capacity of desired neuronal cells, and the spontaneous differentiation (FIG. 23F). To control this bias and generate gene-level enrichment scores, sgRNA representation was normalized in Tuj1-hCD8+ samples to Tuj1-hCD8− samples, the enrichment of the top three guides for each gene was examined, and the empirical distribution of the non-targeting guides was used to normalize enrichment scores (FIGS. 18B, 25G, Table 10 and Experimental Procedures). Top-ranked genes (Table 10) were used to transduce individual sgRNAs to CamES cells and look for signs of neuronal differentiation. Among 20 sgRNAs tested, 19 efficiently induced neuronal differentiation, as measured by the expression of neuronal markers, NCAM, Tuj1 and Map2 (FIGS. 18C, 18D, 24A and 24B). A large fraction of validated genes has been previously characterized to act in early neural development. Examples included neuronal fate-inducing TFs such as Ngn1, Brn2, Klf12, Tcf15, and Mecom. These results were consistent with previous studies showing that the forced expression of these genes induce neuronal phenotypes of pluripotent cells. On the other hand, the function of the remaining hits varied considerably. Major categories included neuronal survival (Jun and Maz), cellular senescence (Sin3b and Rb1), homeostasis/metabolism (Foxo1, Nr4a1 and Nr3c1), and epigenetic regulations (Ezh2 and Suz12). In addition, the neuronal-inducing effects of the majority of hit genes were confirmed via the overexpression of their cDNA in unmodified mouse ES cells (FIG. 24C).
Cells expressing varied neuronal lineage markers resulted from the activation of different endogenous genes were detected (FIGS. 18E and 24D). For example, NeuN and GA BA expressing cells were found for all identified neuronal-fate-inducers. In addition, most hits also induced GFAP and Olig2 positive cells, which indicates the presence of astrocytes and oligodendrocytes. The Glutamatergic neuron marker vGluT1 expressed at varied levels across several hits, such as Zeb1, Brn2, and Nr6a1 (FIGS. 18E and 24D).
It was next tested if these neuronal factors induce transdifferentiation. As reported, Asc11 alone can induce neuronal transition from mouse fibroblasts. cDNAs of individual genes was transduced into mouse embryonic fibroblasts, cultured cells under transdifferentiation condition, and stained them with neuronal marker Map2. Among the 19 genes tested, only Ngn1 induced neuronal marker expression (FIG. 25). However, compared to Asc11, the transdifferentiation driven by Ngn1 was inefficient (7% vs 1% Map2+ cells). All of the other tested genes failed to induced Map2− cells.
Neuronal-Fate-Inducing Activity of CRISPRa To generate a deep view of how sgRNA design and gene activation level affects neuronal differentiation, other high-ranking sgRNAs of the 19 hit genes were investigated. Quantitative PCR results showed that effective endogenous gene activation (10 to 10,000 fold) was achieved by most of their cognate sgRNAs (FIGS. 18C and 24A). It was observed that, for the majority of hit genes, a higher gene expression level generally induced more efficient neuronal differentiation. Outliers of this trend included Jun, Brn2, Suz12, Tcf12, Zeb1, and Hoxc8. Cognate sgRNAs that induced higher expression levels of these genes generated similar amount of neuronal cells.
To investigate the determinants of CRISPRa activation in more depth, the targeting locations of top-ranked sgRNAs of the 19 hit genes was investigated. The observed signal followed a mixture distribution (FIG. 26A) (Horlbeck et al 2016 eLife 2016; 5:e19760). To determine what factors contribute to high neuronal signal, a hierarchical logistic regression mixture model was fit to estimate what genomic features can contribute to or prevent efficient activation (FIGS. 26B and 26C). It was found that KDM2B binding sites, H3K27ac peaks, and H3K4me1 peaks contribute to efficient activation (the top feature CXXC1 was primarily associated with a single gene, FIG. 26D). H3K27ac and H3K4me1 are known marks for areas of primed activation (Calo and Wysocka 2013 Mol Cell. 2013 Mar. 7:49(5):825-37), while KDM2B helps to maintain the stem cell state by recruitment of the polycomb repressive complex 1 (He et al. 2013 Nature Cell Biology 15, 373-384). Indeed, when controlling for other factors, being in a KDM2B increases the average observed log 2 fold change by nearly 1 (0.93, p=0.077, FIG. 26E). On the other hand, it was found that hotspots of open chromatin had little effect of guide efficiency (two-sided t-test, p=0.54, FIG. 26E). These indicated that the epigenetic features of sgRNA binding sites are important for CRISPRa activities.
A Double-sgRNA Screen for Genetic Interactions Driving Neuronal-Fate The strategy to use ES cells differentiation as a tool to discover lineage reprogramming factors was justified by the fact that Ngn1, a hit of the primary screen, is able to convert fibroblasts to neurons. However, as most hits failed to achieve transdifferentiation, the difference between the two processes was highlighted. Compared to ES cell differentiation, a direct lineage programming process utilizes profound transcriptional, epigenetic, and metabolic changes of target cells. These complex mechanisms tend to be initiated by synergistic genetic interactions, instead of a single factor. In most cases, direct lineage reprogramming can only be mediated by the ectopic expression of a gene cocktail. Thus, it was hypothesized that novel gene interactions greatly facilitate direct neuronal reprogramming.
Current gain-of-function techniques, such as cDNA overexpression, are difficult to apply in a pairwise manner, even for a moderate number of genes. In addition, optimal gene expression levels are important for cell fate determinations. Overexpression libraries have limitations owing to dosage and functional issues, and thus may fail to cover genes' optimal expression level. To address these problems, a strategy to determine the gene interactions between the primary hits based on double sgRNA screen was developed. A library of dual-sgRNA-constructs targeting the top neuronal inducers was generated (FIG. 27A). For each hit gene, two sgRNAs were included. These sgRNA-High (H) and sgRNA-Low (L) were validated individually to drive different target gene activation levels (FIGS. 18C and 24A). The double sgRNA construction contains two sgRNAs driven by either human or mouse U6 promoter (FIG. 19B). Thus, two sgRNAs express independently. The library was generated through the ligation of two sgRNA elements, which can be easily scaled up (FIG. 27A). The library also included negative-control sgRNAs, i.e. non-targeting sgRNAs.
With the same strategy as in single CRISPRa screening, double CRISPRa screening was performed (FIGS. 19A and 27B). Pairwise interactions of sgRNAs were enriched relative to individual sgRNAs, and interaction scores were generated for each sgRNA pair (FIGS. 19D, 27B and 27D). It is noted that the correlations between two independent screening replicates are very high (FIGS. 19C and 27C), which indicates high reproducibility.
Hierarchical clustering of sgRNAs based on the correlation of their interactions shows that a fraction of sgRNAs tended to form a high number of interactions (FIG. 19D). These interaction-prone sgRNAs included many that drove low levels of neuronal differentiation compared to their counterparts. For example, Ngn1-H and Ezh2-H, which drove high gene activation and mediated efficient neuronal differentiation when applied individually, did not form strong interactions with other sgRNAs (FIG. 19E). On the contrary, their second top counterparts, Ngn1-1, and Ezh2-L, had synergistic effects with almost all other sgRNAs. A hypothesis to explain this is that in the screening system, some top sgRNAs already trigger saturated readout (neuronal differentiation), thus their interactions with other sgRNAs (even those synergistic) failed to be scored higher than themselves.
On the other hand, for genes whose higher activation lead to similar neuronal differentiation, such as Brn2 and Jun, a targeting sgRNA achieving highest activation tend to form stronger interactions then their counterparts (FIG. 19E). Foxo1-L and Foxo1-H, which mediated quite similar activation activities and differentiation efficiencies, both appeared as interaction-tendency hits (FIG. 19D). All together, these results showed that a library that covers a broad range of induced expression, including a “goldilocks” zone, is optimal for a gain-of-function double screen.
Gene Combinations Identified in Double CRISPRa Screen Convert Fibroblasts into Neurons
Based on false discovery rate, a list of gene pairs that showed strong synergistic effects was identified. Strong synergies included Ngn1+Ezh2, Ngn1+Foxo1, Tcf15+Zeb1, Tcf15+Foxo1, and Zeb1+Ezh2. To confirm these interactions, constructs expressing corresponding single and double sgRNAs were generated, and their effects in neuronal differentiation of CamES cells was tested. All of the identified sgRNA pairs showed additive effects in neuronal differentiation of mouse ES cells (FIG. 19F).
The synergistic links to Ngn1, the top hit in the single guide screen, that was identified have not been previously reported to drive neuronal transdifferentiation from fibroblasts. The ability of the above identified synergistic gene pairs to drive neuronal transdifferentiation from fibroblasts was investigated.
One gene pair, Ngn1+Ezh2, induced over 50% Map2+ cells, which is almost 50-fold more than Ngn1 alone (FIGS. 20A and 20B). Another double screening hit, Ngn1+Foxo1, induced nearly 45% neuronal cells. Zeb1+Ezh2, induced strong Map2 expression on neuronal cells (FIG. 20A). On the other hand, neither is able to mediate neuronal transdifferentiation alone. These results highlighted the power of double screen to discover strong synergies to mediate cell fate transitions.
Here, two new powerful neuronal inducing cocktails were identified: Ngn-1+Ezh2 and Ngn1+Foxo1. It was tested whether the induced cells possess neuron functions. The expression of other mature neuron markers in Ngn1+Ezb2 and Ngn1+Foxo1 induced cells, including Synapsin and NeuN was confirmed (FIGS. 20C and 28A). Furthermore, a large part of induced cells were Tbr1 positive, while a small part was GABA positive (FIGS. 20D and 28B). Moreover, these two combinations also induced neuronal transdifferentiation from tail tip fibroblasts with an extended culture time (FIGS. 20E and 28C).
It was next assessed whether the induced neurons using Ngn1+Ezh2 and Ngn1+Foxo1 were capable of synaptically integrating into pre-existing neural networks. After 7 days' co-infection of cDNAs and a superfold GFP (sfGFP) reporter, the induced neuron cells were re-plated onto rat neonatal cortical neurons that had been cultured for 7 days in vitro. One week after re-plating, patch-clamp recordings from sfGFP-positive induced neuron cells were performed (FIG. 21A), In voltage-clamp mode, it was observed a fast activating and inactivating inward current followed by a slow activating and inactivating current (FIG. 21B). The action potentials could also be elicited by depolarizing the membrane held at −75 mV in current-clamp mode, which could be inhibited by the application of 100 nM tetrodotoxin (TTX), a selective blocker of voltage-gated sodium (Na+) channels (FIG. 21C). Inward currents could be blocked by the application of 500 nM TTX (FIGS. 21D and 28D), and outward currents could be inhibited by the application of 5 mM tetraethylammonium (TEA), a selective blocker of voltage-gated potassium channels (FIGS. 20E and 28E). Together the voltage-clamp studies show that these induced neurons express functional voltage-gated Na+ and K+ channels, which are critical in the ability of neurons to fire action potentials.
For all the induced neuron cells analyzed (5/5), action potentials that fired spontaneously were observed (FIG. 20F). Application of 100 nM TTX blocked the spontaneous action potentials, and washout of TTX completely reversed the blockade (FIGS. 21G and 28F). Spontaneous postsynaptic currents were recorded in induced neuron cells held at −60 mV in the voltage-clamp configuration. These currents could be blocked by application of 30 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an AMPA and kainate receptor antagonist (FIGS. 20H and 28G). The blockade is reversible upon removal of DNQZ. On the contrary, the presence of 30 μM Bicuculline (BIC), a GABAA receptor antagonist, slightly increased the observed frequency and amplitude of the spontaneous postsynaptic currents (FIGS. 20I and 28H). These experiments demonstrated the induced eurons were mostly glutamatergic excitatory neurons, which fired AMPA/kainate receptor-mediated spontaneous excitatory postsynaptic currents (EPSCs). The emergence of AMPA receptor mediated synaptic transmission is a key step in the development of mature glutamatergic synapses (Wu et al., Maturation of a central glutamatergic synapse. Science. 1996; 274:972), These data indicated that these induced neurons can form mature neurons as they form electrically active networks of cells in vitro. Overall these experiments demonstrate that functional synapses can be formed with induced neurons using Ngn1+Ezh2 or Ngn1+Foxo1.
FIG. 29 shows three additional powerful neuronal inducing cocktails: Ngn1+Brn2, Brn2+Ezh2, and Mecom+Ezh2; which could drive neuronal transdifferentiation from fibroblasts.
Table 13 Shows Exemplary sgRNAs for Genes Targeted in Examples 1-4.
TABLE 13
SEQ
gene guide ID
6430411K18Rik GTTGCTGGTTGATGAAGTTG 586
6430411K18Rik GCAGTTCCAAGTACCGGTGC 587
6430411K18Rik GGAAGGCAGCGCCATTCTGG 588
6430411K18Rik GACTCAGAGGACCCAAGAAA 589
6430411K18Rik GGGTCGAGTCCAGGATGAGT 590
6430411K18Rik GGTGGACTTGCTTGCAGGGT 591
6430411K18Rik GGATGATGAAGAAGAGGAGG 592
6430411K18Rik GCACACACTCCGACTCATCC 593
6430411K18Rik GGCTCCTTGGCACAGTACTC 594
Adipoq GAACCTGGTTTAATCCAGCT 595
Adipoq GGTAGAGAATGGCCAAAGCC 596
Adipoq GTCCCATATAGGAACACTGC 597
Adipoq GTTTCTAGAGAAATCACGTT 598
Adipoq GCTGGGTCTGGTAGACACCC 599
Adipoq GAAGCCAGAAGCCAGTAAGA 600
Adipoq GTGAAGACCACGAGGCATTG 601
Adipoq GTACAGGAAGGTTCCTGGTG 602
Adipoq GGAGTCTTAAGGCAGCTGCC 603
Aebp1 GATGTCACTTCCCTAGGCAT 604
Aebp1 GGCACAGCGGGTTAGAGCAC 605
Aebp1 GAGCACTCAAAGGGTCCAAG 606
Aebp1 GAGGGATCACACAACAGCAC 607
Aebp1 GTCATACTTGGACTGAATTT 608
Aebp1 GAGTGGAGAGCTCTCCTCAC 609
Aebp1 GGAATTCGAGCAGAGGAGCT 610
Aebp1 GACAGAGAGGGTGAGGGTGA 611
Aebp1 GGTGATCGCCAGTACCCTCG 612
Aebp1 GGATGTCACTTCCCTAGGCA 613
Aes GCCTGGACACCCAGGCTTCA 614
Aes GAGGAAGCCTTAGAGACTGC 615
Aes GATTCTGGTATCCCGGAGGC 616
Aes GGCCTCTGATTCTGGTATCC 617
Aes GAGTCTGTGGCCTTGGGACT 618
Aes GTCTCTGTCTGTCTCAGGTA 619
Aes GACACCTGTCCCACAGAGGT 620
Aes GGATGGGACACCACTGAGGG 621
Aes GACTCAGCAGCTTAAGAGGA 622
Ahr GATGAGAAGGAAAGAAGCAC 623
Ahr GCCCAAGCAGAAATGAGATC 624
Ahr GTTGAGTGCCATGTAAGTTA 625
Ahr GCCTTCCTTGTTGAAATAAC 626
Ahr GCAGAGATGATAAAGGAAGA 627
Ahr GGAAATGACAACAGGAAAGT 628
Ahr GATTTAATGGGAGTGATGAG 629
Ahr GTCATCACGTGCTGCGAAGA 630
Ahr GTCCTTTAATAAGGTCTTCC 631
Ahr GAATGTGTATGCCCTGTGAT 632
Ahrr GGGAAGCTCCTGCTACCCAG 633
Ahrr GTGTGAAATACCTTAAGAGT 634
Ahrr GTCAGAACCTTGCATAGATG 635
Ahrr GAGGCATCTGGAAGTGCAGA 636
Ahrr GGATTTGGTGCACAAACTGG 637
Ahrr GTGCCTAGGTGGAAGGTGGG 638
Ahrr GGTGGGAGGGACTGGATGAG 639
Ahrr GGGTAGCAGGAGCTTCCCAG 640
Aire GTACAATCTCACTTTGCTGG 641
Aire GCACCACGACACCCAAGGAA 642
Aire GGGCCCAGCTTTCGAAAGCT 643
Aire GAACAGGGAGCAAGGGACTG 644
Aire GCTTGGAGGCCCTGTCTTTC 645
Aire GAGATTCCTCACTGGCATGA 646
Aire GTTTAGCCTAGAGCCAATCA 647
Aire GGTCAGTCACTTCAGAGCCG 648
Aire GCTAGAGACTGCCCTGCCTT 649
Alx1 GCGGCTGTTAACCGGCTTGC 650
Alx1 GGGCACAAGGCCAAGCAGAA 651
Alx1 GCATCCGACAGCAAACGAGA 652
Alx1 GACAGCAAACGAGAAGGCCA 653
Alx1 GGGAGTCAGGGCTCTAAGAT 654
Alx1 GTCGAGGCGACTACGATTCT 655
Alx1 GCAGAACTGTTAAGTGAAGT 656
Alx1 GTTGCTTGCTCCAcCTTCTC 657
Alx1 GACAAATGCCAGGAGAGACA 658
Alx1 GAAGCTTGAAATAACAGGCT 659
Alx3 GAGAAGAGAGGCCTCTACTG 660
Alx3 GAATGGAGAGTCTTGTAGGG 661
Alx3 GCTGTAAATCAAGGCCAAAC 662
Alx3 GACTGCAGGCTAGGCAGAGA 663
Alx3 GGTTTCACAGTGGTCTGCCC 664
Alx3 GAATGTTGGAGGAGGGATGG 665
Alx3 GTCCTTGGTTGAGGGCAGTC 666
Alx3 GCCATAACACTGTTTCTGAT 667
Alx3 GCTTAAAGATCCCTTAGGTC 668
Alx4 GTGAGGAGAATTCCAAAGAA 669
Alx4 GTTAGCTTTGAGGTCTCCAT 670
Alx4 GTTGAAGCAAAGGTCACCAA 671
Alx4 GGATGAGAGGAGTGGGAAGA 672
Alx4 GAAACCTGTGTCTGTCTCTC 673
Alx4 GCTGGAGCAGATTGGAGGTA 674
Alx4 GAGATAGGTGAGATTGGAGG 675
Alx4 GATTCGACCCGGAGAAGCCT 676
Alx4 GGAATTTCAACAGTGTGGTG 677
Alx4 GTCAGCATCTGGATGCCTGA 678
Alyref GTTCCCTAATGTCTAATTAC 679
Alyref GACCAATCGCCGCTCGCTTC 680
Alyref GAACTGCGGCATCTGCAGGA 681
Alyref GAAGCGAGCGGCGATTGGTC 682
Alyref GCCCAGCAAGCATGACAATA 683
Alyref GGCACACGCCTCTAATCCCG 684
Alyref GTCTTACCTCTGTAGCATCC 685
Amer2 GTGTAGGGAAGGCTCCTTGC 686
Amer2 GCGTTCTAAATCAACCTGAG 687
Amer2 GACAAAGCAGCTTTCAGTGT 688
Amer2 GCATTGTTCTTTGTGGACAT 689
Amer2 GAAAGAGGAAGACTGAGCCC 690
Amer2 GTGAGAGAGAGCAGTTTCCA 691
Amer2 GTATTCTTTCTCCTCTGTGG 692
Amer2 GCTAATTGGTATTTGACTGA 693
Amer2 GAGAACAACCTGTGTGGGTA 694
Ap2b1 GTTTCCCTGTCTCAGGGATA 695
Ap2b1 GGGTGCGCGGGAGAACCAAA 696
Ap2b1 GATCTCCAAACCTGATGGTC 697
Ap2b1 GGCTACCTGGCAGTGAGGAA 698
Ap2b1 GGGCTGGAGAGATGGCTCAG 699
Ap2b1 GGTTTCCCTGTCTCAGGGAT 700
Ap2b1 GGAGAGATGGCTCAGTGGGC 701
4p2b1 GGTCAAGATTTCCTGATTAA 702
Ap2b1 GGCTGGAGAGGTGGCTCAGT 703
Ap2b1 GATCAATCATGGTTAGCCAG 704
Ar GCCTAGTCAGCTCCTGGAGA 705
Ar GGCTTTAGAGAACGTAGTGC 706
Ar GCACAGAGGTAAACTCCCTT 707
Ar GAAACTTCACCGAAGAGGAA 708
Ar GGGTCTACAACCTTTCTCTA 709
Ar GAGTTAACTGAAACCTCAAG 710
Ar GGAGTTAACTGAAACCTCAA 711
Ar GCCCACCAGGACAAGCAGAA 712
Ar GCGTCCCTTAAGCTTCTGTA 713
Arf2 GCTGGTATGTGGGAGGAGCC 714
Arf2 GACCAATGGAAATGGCAATA 715
Arf2 GGGCTCTGGTAGGAGATTAC 716
Arf2 GATTGGTCGTCTGTGGCTTC 717
Arf2 GCAATGGTATTGAAGAGGCA 718
Arf2 GCGCAAGAGTTCCCAGGAGG 719
Arf2 GAGGCTTTGGGAGACTGCTA 720
Arf2 GTAGGAGATTACTGGAACTC 721
Arf2 GAATCTGGGTATTTCTGACC 722
Arf6 GCTTCGTCGGCCCTTAGGAC 723
Arf6 GAAGTCAGTGAAAGGGAGCA 724
Arf6 GCTAGTTACTGAAGACGTTC 725
Arf6 GGAACATGGCACCTGACCAG 726
Arf6 GAGTTTAAACTTTCAAAGGC 727
Arf6 GTCTGTTTCTTAAGAAATGC 728
Arf6 GCAAGGGAAGGTGACAGAGG 729
Arf6 GAGAGGCAGGTTGTAAGTGG 730
Arid3a GCAGGGATATATTTAGCCAA 731
Arid3a GGACCTGAGCACCACCTATG 732
Arid3a GTCCTGGGAAAGCTTGGAAA 733
Arid3a GAGGTGGTGGGTGTCTCTCC 734
Arid3a GTCCCTTGTTAGACTGTTGT 735
Arid3a GAACCGTGACGACCGTACCT 736
Arid3a GCTCCTAGGTACGGTCGTCA 737
Arid3a GGGCTTCAACCCAGCAGTGG 738
Arid3a GGAGAAGAATGCTGGTGTGC 739
Arid3a GTGACTTTCCGCTCAGAGGT 740
Arid3b GCCTAGAGAACATTTATACT 741
Arid3b GAGGAGGGACAGGCAGTAAG 742
Arid3b GTTACATCTCTAGAGCAAGG 743
Arid3b GAGACGCGGGCTAGTGAAGC 744
Arid3b GTCCGTTGCTCTCGGTTTGG 745
Arid3b GGTAAGGGAAATGGTCACCA 746
Arid3b GCTTTCCTCAGCAAGGGAGA 747
Arid3b GTTTGCCATGGTAGCACTTA 748
Arid3b GAGGACCTGACCAGGGAAGT 749
Arid3b GGCAGCGGCTTTCAGCAGAT 750
Arid5a GTTCGCAGGTTGCCCGAGAC 751
Arid5a GCTAGAGTCTTGGATCTCTT 752
Arid5a GAACCGGCCAGGACCACTTC 753
Arid5a GAAGTATGGTCACTGTCTCC 754
Arid5a GAAATTGTCCCTTGGTGATC 755
Arid5a GGATTAGCTGTGGCTTTGAA 756
Arid5a GGCTGTGTCCCAGATCACCA 757
Arid5a GTAGCTTGCCAAAGACTGGG 758
Arid5a GCAGATCTCCATACCTAACC 759
Arid5a GGATGGAGAGTGATGGAGGG 760
Arid5b GTCTGCTCGGAATATGAATT 761
Arid5b GTGATGTGCAGGTCATAATT 762
Arid5b GTATATTATTCCTGTAGCGC 763
Arid5b GCAAACCGCGCAATGCTCCA 764
Arid5b GGCACCAATCTTTCCAGAGT 765
Arid5b GATTGCATCAGGTCCTGGCA 766
Arid5b GAGGTTTAATACACAATCCA 767
Arid5b GAAATATTCAGAGCTGGGTT 768
Arid5b GGCCTATCCGATACTGAGAA 769
Arnt GTTTGAAACTCCAGGTTAAT 770
Arnt GTGTAGTGGAGTCGTCTTTA 771
Arnt GAAACAGTAAGTCGCCATAG 772
Arnt GAGTTGGCTCTGAAGCTGGT 773
Arnt GTGAGCCGACCAACTGGAGT 774
Arnt GACTGACCGCGCCCATAGTT 775
Arnt GGATTAGGGAAACAGCTGGT 776
Arnt GCATTTCACTGACGTCAATT 777
Arnt GGCCGGATTAGGGAAACAGC 778
Arnt GCGTGTCTTCTGCCCAGGAT 779
Arntl GAGAGATTCCTTCACAGAAC 780
Arntl GACGAAGTGGCCTTGCTATC 781
Arntl GAGGAGGAGGGAGAGCTGAG 782
Arntl GGCTTCTCCTTGTGCAAACC 783
Arntl GTGCCAATTGGTCCACTCCT 784
Arntl GGTGCCAGTAGAAGATAAAC 785
Arntl GTGGAGCTGICATTCCCGAT 786
Arntl GGACCAATTGGCACGCTCTG 787
Arntl GATAAATTCATTGTTCTGGA 788
Arntl2 GGGAGCTTCATGTGCAGAGT 789
Arntl2 GCAGCCTCACTTCCTGGCTC 790
Arntl2 GCTGCTGGTGTCTGAGGAGT 791
Arntl2 GACAACACCATTCAGTTGTT 792
Arntl2 GGGTGTTCATTTATTTCTGG 793
Arntl2 GCAGTCTGGAAGCTCAGGGT 794
Arntl2 GGTCTGGAACCGGTTGGAGG 795
Arntl2 GGAGGATGCTATTGATGGGT 796
Arntl2 GGTGGGTGTTCATTTATTTC 797
Arntl2 GGGATCGGTGAGAGAGCAAT 798
Arx GAGGTCCATTGGTCCTAGAA 799
Arx GGGAATGAGGGTGTCCATTC 800
Arx GGCAGAGTGAATATTAAGTT 801
Arx GAGTATTCAGAGAGGTGAAA 802
Arx GGAGTCCTCAACGCAACTTG 803
Arx GACCCAACTTCACTCAGGGT 804
Arx GTCCTCAACGCAACTTGAGG 805
Arx GAGGGAGGTGGGTAAGAGGT 806
Arx GATGGTTGCCTCTGACACGT 807
Ascl1 GTTGTTGCAGTGCGTGCGCC 808
Ascl1 GTTCCCTAAGAAGCTGAGGC 809
Ascl1 GAGGCAGGAGAATAAGTTGG 810
Ascl1 GATGTTTGAGGATGACGTCA 811
Ascl1 GAGGGAAAGGCTGCTCAGAC 812
Ascl1 GGGCACAACTCGCTAAGGGT 813
Ascl1 GCCTGAGACAGGGAGGGACA 814
Ascl1 GCTAGACGCTATGGGAAAGG 815
Ascl1 GAAGCAGAGACTGTGGAATG 816
Ascl2 GCAGTGTGTATGGAGGTTGG 817
Ascl2 GAGCATGTACTGCCAGTGTG 818
Ascl2 GATTGTATTCTCTCAGGTCA 819
Ascl2 GGTGACAGTTCCCTAGGGAT 820
Ascl2 GGGAGGAAACAGGGCAGCAG 821
Ascl2 GGAGCATGTACTGCCAGTGT 822
Ascl2 GCTGGCTGTAAGGTGCAGAT 823
Ascl2 GCACCTACCTAGTCCTTTGA 824
Ascl2 GCCAGAAGGAAGCGTACGCC 825
Atf1 GCTGGCCTGTTGTTCAGGCC 826
Atf1 GTGGAAGTGCTGGATAAGAA 827
Atf1 GAACTATCTGAGAGGATCCC 828
Atf1 GTGACCTACAAAGTAAGGTC 829
Atf1 GCTTGGAGATAGGCTGGCTG 830
Atf1 GGACTTAGCATGTCCTTGTG 831
Atf1 GATAGCGACTCCAGAGAGGT 832
Atf1 GCCAAGGTCAGAGCATGGAT 833
Atf2 GGCAACACCCATATTATCTC 834
Atf2 GCTTCTCGGTACACGGAGAG 835
Atf2 GACCGTTGTTTCGGTAACCA 836
Atf2 GAAGAAAGCGGCAGGGATGC 837
Atf2 GAGAGAAGAAAGGTGAGGTC 838
Atf2 GGACCGTTGTTTCGGTAACC 839
Atf2 GAGGGAATAGACCTGGTGTT 840
Atf2 GTCAGCTGCTCATTACTGGT 841
Atf2 GCCGACAATATCTAACCCAA 842
Atf2 GGGAAGATCGGCTCCAGTTC 843
Atf3 GAAGGAAGAGCCCTAAGGTC 844
Atf3 GACAATCTCCCGCGTGAAGG 845
Atf3 GACCGGAGCTGATCTGCATA 846
Atf3 GGGATTACAGCAGCATCGCG 847
Atf3 GGGTTGTGGAGGTGTGGAGC 848
Atf3 GCGCAGGGATAAGAAAGGGC 849
Atf3 GCCTTGGACTTGAGGAACCC 850
Atf3 GGGTTTACCTGCCGGCAGGT 851
Atf3 GATCTGCATACGGGCTCCCG 852
Atf3 GGAGGGCAGAGGCCTGTGAA 853
Atf4 GTGAGTCACTTAAACAGAAG 854
4tf4 GGAACTGACCCTATACAAAC 855
Atf4 GGACTTGGCCTCAGAGACCA 856
Atf4 GTCCCTGTCCCAGGACTGAC 857
Atf4 GAGGCCTTGACCAGTACCTG 858
Atf4 GGCAGTGAGGGCCTCTATGA 859
Atf4 GCAGAGCCAATAGGAACTTG 860
Atf4 GGAGGAGCCACCAGAGGTTC 861
Atf4 GAGGAGCCACCAGAGGTTCC 862
Atf4 GGCATAGGAGGTTAGACCTG 863
Atf5 GGGCTTAACCCACGAGGTCT 864
Atf5 GGACACACAGAACGATCATA 865
Atf5 GACTTAACAAAGCCCATAGC 866
Atf5 GGCCTCTAGGGACTTGCTAG 867
Atf5 GCTACCTAGGAGCTGTTGCC 868
Atf5 GAGGCCTCACAGGACAGGGT 869
Atf5 GGAGTTGTGATCATCCCTGG 870
Atf5 GAGAGAACAGCCTTGTGTGA 871
Atf5 GTCCCTCTTGTCCTTCACCG 872
Atf5 GCGCATGCGCAACAGGTTGT 873
Atoh1 GCGGAACATTTCAACGGCAC 874
Atoh1 GAATTTCCAGAACTGACTAG 875
Atoh1 GACAACGTGAGAGCCTGGAA 876
Atoh1 GCTTGGAGGGATCCCAGGCC 877
Atoh1 GCTCAGATGAGACCCAGGGT 878
Atoh1 GCAATCCCATGGACACGCTC 879
Atoh1 GCCTGAGATCCCTCCAAGCC 880
Atoh1 GAGAGCACTAGGAGCAAGCT 881
Atoh1 GTTGAAATGTTCCGCTAGCA 882
Atox1 GAGTGGTATCAGTTCCCTTT 883
Atox1 GATGGCCAATTCCAGTTCAC 884
Atox1 GGACACCAAAGCTGCGCTTC 885
Atox1 GTCATTTCTGAAACAGGGCT 886
Atox1 GGATTCACATCAGCTCCTTC 887
Atox1 GTGGCTCTTGCATCAGCCTC 888
Atox1 GCTAGGTTCCTCCCTTGGGC 889
Atox1 GTTCTGGCTGGGAGGCTTCT 890
Atox1 GTGCAGATTAAAGTCATGGC 891
Bach1 GCGAGCTCCGTGTAACGTTG 892
Bach1 GTAGCCCTGGCAGGACTTGT 893
Bach1 GCCTTCAGCAGGTGAGGAAG 894
Bach1 GCACTGTCTGTGTGTGTTTA 895
Bach1 GCTTATTCCATGCTATTCTA 896
Bach1 GCACCAGGTCACCACTTACA 897
Bach1 GAAGCTAGTGATCATTCAGA 898
Bach1 GCTCTTACTAGCGGAGGGCG 899
Bach1 GTGGTTCTGACAGACATTCG 900
Bach1 GTGGTCTACCAGGCTGTGAG 901
Bach2 GAGGCAAAGACCGGAGCTCT 902
Bach2 GGTTGTGTTAGTTGCTGTGC 903
Bach2 GACTGAAATTGACCTCTACT 904
Bach2 GAAGGCCAGTGTGGCACGTG 905
Bach2 GTTTGTCCTTTGTTGCAATC 906
Bach2 GGCTGGAGCAACACTTTGGA 907
Bach2 GAGAAACACTAGAACCACTG 908
Bach2 GCATGTGGCATTGCTAGCTT 909
Bach2 GGGCCTGATTCAGCTTTCCA 910
Barhl1 GTTGCAGCTACTTGGAGACC 911
Barhl1 GGATCAGCCACTGCTAGTGC 912
Barhl1 GGAGCTGCTAGGAACCCTTG 913
Barhl1 GCCCTAGAGAGGCCACTGAC 914
Barhl1 GGGTCCTAAGAGGTTGGGTT 915
Barhl1 GGAAATTAGGAGGAAGAAAG 916
Barhl1 GTTGAGCCACACACTCACCC 917
Barhl1 GCACAGACCTAACTATTTAC 918
Barhl1 GTTGCGCATCTGGGCAGCAG 919
Barhl1 GAGAATTGTGGTGTTCTACA 920
Barhl2 GAGCAGACATTTATTTATCA 921
Barhl2 GTAGTCTTTGGCGCCAGAAC 922
Barhl2 GCTGACGGCACAGCTTGTGC 923
Barhl2 GAGTAGAGCTCAGACGTTGC 924
Barhl2 GAGTGCTATCTAGATGGTCT 925
Barhl2 GGATGACAAGCCAACGCGCG 926
Barhl2 GACCAAGGCCTAACCTGGGA 927
Barhl2 GTCGCGTTCCAGGTCCCAAA 928
Barhl2 GCGCGTTGGCTTGTCATCCG 929
Barhl2 GGGATGGAAGCATTGGAGGG 930
Barx1 GGCGCACCTGTGGAATGGAG 931
Barx1 GAAACTGGCCGCTCTGGGAG 932
Barx1 GCTCTGTGGAGAGCATTCGT 933
Barx1 GAGCTGTAGCAATGAGCTTT 934
Barx1 GGAATGCCTGAACCAGTCCT 935
Barx1 GCCTGATGTTGAGCCCTCCA 936
Barx1 GCGCATAGTGTTCAAATACA 937
Barx1 GCACCTGTGGAATGGAGTGG 938
Barx1 GGGATCCAGTGCAATACACC 939
Barx1 GTGAGTAGAAGCGGCTTTCT 940
Barx2 GGCGCTAAGGGAGGACAGAG 941
Barx2 GAAAGTTTGTAAGGCACCGA 942
Barx2 GCTGTGGGCTGGGTTGAGAG 943
Barx2 GGCAATCAAGTTTGCAACCT 944
Barx2 GCTTGCGCACACCACTTCAG 945
Barx2 GCATTTGTGAACCCGTACCC 946
Barx2 GCGAGTACCAACGAGGGAAA 947
Barx2 GCCATGAACACATGATTGTA 948
Barx2 GTATAACACACTTCTGGTAT 949
Barx2 GCTCCGAGCATGGTTAGCCG 950
Bbx GTGAAAGACACATGGCAAAG 951
Bbx GTCAGTGGGACCTGACCGTG 952
Bbx GTCCAATTAGTGTTAATGTC 953
Bbx GATCCCTTCTGCACTGAGTT 954
Bbx GCCCATATCCACGTGGACTA 955
Bbx GAGGCAGGGACAAACCAGGT 956
Bbx GACTGGGACGTGAGAGCACA 957
Bbx GAAAGGTAACAAATCAAACA 958
Bbx GCTACTTAGTCTTT3AACTA 959
Bbx GCTCAACACCTGGGAACTAA 960
Bcl6 GTGGGAAGAGAGAGAGAGAA 961
Bcl6 GTGGCTCGTTAAATCACAGA 962
Bcl6 GCTCTGTTGATTCTTAGAAC 963
Bcl6 GTCCTTTCTTCTCTCTTTAT 964
Bcl6 GGTGGGAAGAGAGAGAGAGA 965
Bcl6 GGTAGAGCCAGCCAGAGTGG 966
Bcl6b GGCCTCTCCCTTCTGTTCTT 967
Bcl6b GGTCGTATCGTGGATGGCTT 968
Bcl6b GTCTGCATCCTTCCACGAGA 969
Bcl6b GCGGAGGGTGGTAATATGGG 970
Bcl6b GCTGAGAGCTTGATTGATGG 971
Bcl6b GAGGTTAGTGGTGCGGAGGG 972
Bcl6b GAAGCTAGGAGAGGATCTGA 973
Bcl6b GCCGAAGAGAGCAGGGACCT 974
Bcl6b GAGAAGGAGGAGTGATTGAC 975
Bcl6b GCATGAGTACGCAACTAATT 976
Bhlhe41 GCATTTAGCAGGAAGAAACG 977
Bhlhe41 GGTTCCTCGAGTAGGACGAC 978
Bhlhe41 GATAAGCCACGCCGAGAGTG 979
Bhlhe41 GGCTTCCTCCAGTTCTTAAC 980
Bhlhe41 GAGCAATTTACACCTTGAGC 981
Bhlhe41 GGCGAGCCCACGTTTACTAC 982
Bhlhe41 GAGACTCAAGTTTAAGGCAG 983
Bhlhe41 GGAGGTGCCAGTAGTAAACG 984
Bhlhe41 GGCTTATCGCACGAGGGAGA 985
Bhlhe41 GGAGACAGGATTAAGGAGGG 986
Bmp7 GGCACTTCCTCCTAAAGTCT 987
Bmp7 GGCCAGGGACTCAGTACTGG 988
Bmp7 GTGCTTCTGTGGTGGGAAGA 989
Bmp7 GCGTGTTTGTTCTGTCACTT 990
Bmp7 GACTGGAGCAAATGGAGTGT 991
Bmp7 GTCCAGCACCCAAGGGATCC 992
Bmp7 GTCTCTCTGTGGAGACTCAG 993
Bmp7 GTTAGACAGTGAGGTACCAA 994
Bmp7 GCACCGAAGAAGGGAGAGAT 995
Bmp7 GGCAAGCATGGCAACCTCCA 996
Bmyc GAACTGGGTCCAGATAGGAA 997
Bmyc GGCATGATAGCCCAGGAGCT 998
Bmyc GTTGGTCTGCTCACATGTTA 999
Bmyc GTGCAAAGCCCAGTGGAGGC 1000
Bmyc GAGCAGAATTCCAGCAGGAC 1001
Bmyc GGTCCTGCCTCCAAGTGTCC 1002
Bmyc GGACACTTGGAGGCAGGACC 1003
Bmyc GATACTTTGAGCTTGGCGTC 1004
Bmyc GAGCCTGGTGAAGGGACTGT 1005
Bnc1 GGGTGCAGAAATATGTGGAG 1006
Bnc1 GAAGCAGGTGCAACAAATTG 1007
Bnc1 GCCTTCTGCCIGGTCCACAC 1008
Bnc1 GGGACTGGCTGTTTGGGTCT 1009
Bnc1 GGCTGTTTGGGTCTTGGGTC 1010
Bnc1 GACCCAGAACAGGCACCTGG 1011
Bnc1 GAGAGCAATGTGAAGGGCCA 1012
Bnc1 GGTGACTTCGCTCTCAGAGT 1013
Bnc1 GAAAGGACTCAGAGACAAGA 1014
Bnc1 GTGCCTGTTCTGGGTCTACC 1015
Bptf GCGAGAGGGAAGAAACAAGA 1016
Bptf GGTTTGGGAGACACGCATTG 1017
Bptf GAGGTGTGGAAGTTCGTAGC 1018
Bptf GGCTCAACACAGGGTCCTCG 1019
Bptf GTTGTTCCAGGAATGCACGC 1020
Bptf GTCAGGTAGACATTATTTCC 1021
Bptf GAGTGGTAAACTTACCCACA 1022
Bptf GAACTTAAGGATAGGAAGGA 1023
Bptf GCCTTTGGGAGCTCTCTATT 1024
Bsx GAAGAGACTGAATGTCACTT 1025
Bsx GTGGCTGGGAACCIAGACAT 1026
Bsx GTTATGGGTAAGGGTGGCGG 1027
Bsx GCGATTCCTCGAGCAATCTG 1028
Bsx GTGTTCCTCTTTGTCTGGAA 1029
Bsx GCCTGGCAGCAGCCAGTGAA 1030
Bsx GTGAGGATCTACACAGTGGC 1031
Bsx GAGGCAAGAAGACAAGCGCC 1032
Bsx GGGAGCCCGGCGAACCAATA 1033
Carf GGCCTACAAGATATCTCAGT 1034
Carf GTCACTCAGAATAAAGAAGC 1035
Carf GGTACTTGATGTGTGCGGGC 1036
Carf GAACGAGTCGGAAGGGAACT 1037
Carf GTAGTTGTAGATGAGGAATC 1038
Carf GGAAAGGAGAACGAGTCGGA 1039
Carf GCATAGTCCCATTTGTACCA 1040
Carf GAGCTGAAGTGCTTGTGTCC 1041
Carf GTCAACTGGACAATTAATGA 1042
Cav1 GGTGCAAGGAAGAAGCACGG 1043
Cav1 GGCACCTTGGAGGAATGGGC 1044
Cav1 GCTCTGGAATCATAAAGATT 1045
Cav1 GCCTCTTGGCTGTTCGCCAG 1046
Cav1 GGCTTTCCCTCTGCTGGTTT 1047
Cav1 GAGAAGGAATACAGAGGAGG 1048
Cav1 GCCTCCTTTGTCTTATTGTA 1049
Cav1 GGCGGTGGTACTTGTGAGGG 1050
Cav1 GAGAGTGATCTAAGTAAGGG 1051
Cbfb GGGAAAGGAGAAACAGAAGT 1052
Cbfb GTAAGCATCTAACCAAATCA 1053
Cbfb GCGCTAATTGTTTCTCATAT 1054
Cbfb GCTGATACAGACCACTCAGT 1055
Cbfb GGCTGATGCTAGCGTTTGCC 1056
Cbfb GAACAGATTAGGTGCATGAA 1057
Cbfb GAGGACTTTGCATACAGGGA 1058
Cbfb GTGGACTGTGCACTGAAAGG 1059
Cbfb GAAGTGCTGAGGAAGGAGCA 1060
Ccnt1 GAGCTTCGTTTGAGTGTTTG 1061
Ccnt1 GGATCTCCGGTAGAACGGAA 1062
Ccnt1 GAGATGCTGATACAAGACTA 1063
Ccnt1 GAACTACTGACCTGACGCGC 1064
Ccnt1 GGTGGAGGAGAAAGGATCTC 1065
Ccnt1 GACGTGACGAACTTCCTCCA 1066
Ccnt1 GGTTCCTGGGTTCTTAGCTC 1067
Ccnt1 GTAGACATTCTAAAGAGAAG 1068
Ccnt1 GTGTGGCAAGGCACTGAGCA 1069
Ccnt1 GTGTAGACATTCTAAAGAGA 1070
Cdkn1c GGGAGTCGAGAAGGTGACTC 1071
Cdkn1c GCTAACCAGGTCCAAGGTCG 1072
Cdkn1c GCACACTGCTTCCAGAAGCA 1073
Cdkn1c GAGGAACAGAATGAGGGCTG 1074
Cdkn1c GTCTGTTGCGAGGAGGAAAC 1075
Cdkn1c GAAGTACCCATTCTGCCCAA 1076
Cdkn1c GGTCCAAGGTCGAGGTCCCA 1077
Cdkn1c GGGCTCCTTTGTCTGCAGGC 1078
Cdkn1c GGAGTGTGGTCCTGTGACCA 1079
Cdkn1c GAGTTGGTTCGAAGAGCTGG 1080
Cdx1 GATCCTCGTTGGTAATGGAA 1081
Cdx1 GAGTTCTGCCCTTTCCTCTC 1082
Cdx1 GCCAAGCTAGAGAATTCTTT 1083
Cdx1 GGCGGTATGTCCACCCTTTG 1084
Cdx1 GGTAGTGGCTTAGAGATGGA 1085
Cdx1 GTAAGGTAGCGGGCGTCTCT 1086
Cdx1 GGTTCCGTCTGTAAGGTAGC 1087
Cdx1 GAAGGCCTAGCATGGAGGGC 1088
Cdx1 GCCTGCCTGCCTGTCTTCAA 1089
Cdx1 GACGCCCGCTACCTTACAGA 1090
Cdx2 GAAATGATACTGACAGGAAC 1091
Cdx2 GGGATGTGAAGGGTGGAAGG 1092
Cdx2 GCAGTAATGAATAGCGACAA 1093
Cdx2 GCATTCGGAAGACACAGGCT 1094
Cdx2 GAGAGCATTGTCAGCATCCT 1095
Cdx2 GAAGCTCGTAGCTAGCAAGA 1096
Cdx2 GTCTTTGAACCTGTGATTGG 1097
Cdx2 GGTGAGTACAGTAGCTCTGT 1098
Cdx2 GAGCTTCCTCCTTCCAACCT 1099
Cdx2 GCACTTTAACCTCCAATCAC 1100
Cdx4 GTACATAGATGAGCAAGAGA 1101
Cdx4 GGTCAATTACTCTTGAGTGT 1102
Cdx4 GAAATGAGCAAGTGTCATTG 1103
Cdx4 GAGCAGACTGCTCCTGCTCC 1104
Cdx4 GAGTATGCGGCTCAGAGCAA 1105
Cdx4 GAAAGGCAGGCCTCAGTGAA 1106
Cdx4 GGTAGCCAGGTCACAACACA 1107
Cdx4 GTCCCTGAAGTGGCGCTGAT 1108
Cdx4 GCTGATGGGCTAGGAGCTAG 1109
Cdx4 GTCATTGTGGTGGACCTGCA 1110
Cebpa GAGAGACGTGGGTGCTCACC 1111
Cebpa GCAGGTTTGTTTACCTGGGA 1112
Cebpa GCTGGGTAGCAACGTCTGCC 1113
Cebpa GTGAGCAGAGGATCGCTCTC 1114
Cebpa GGTCACGGAACACGGACAAA 1115
Cebpa GATCGAAGGCGCCAGTAGGA 1116
Cebpa GTGACTTAGAGGCTTAAAGG 1117
Cebpa GGAAAGTCACAGGAGAAGGC 1118
Cebpa GTGCTAGTGGAGAGAGATCG 1119
Cebpa GACTTRCCAAGGCGGTGAGT 1120
Cebpb GGTCCCTGAACTGGCCTCTC 1121
Cebpb GGAGAAAGTCTCCCAAGCCT 1122
Cebpb GAAATGTTGGCAGGAAGCTA 1123
Cebpb GCCTATTGAGCAAAGAACCT 1124
Cebpb GTGGCCAGACCAACCAAGAA 1125
Cebpb GCTCAGAGACAGCAGAGGGC 1126
Cebpb GTCATTTCTCCAGCTCTTGG 1127
Cebpb GGCTGCAAAGGTCTCTGGTG 1128
Cebpb GGGTTCTGCCACACTGTGTC 1129
Cebpb GATCTGTTTCCCAAGAGTTG 1130
Cebpd GTTGTGTTTACAAGACAGCG 1131
Cebpd GAACCACGGTTCACTAGTTC 1132
Cebpd GATGCTATGCTACCACCAGG 1133
Cebpd GAAACGCACCGCGGTTAGGG 1134
Cebpd GCTCCTACCTTCAGTTCCTG 1135
Cebpd GCCTTCAGACATAGCAAAGG 1136
Cebpd GACATAGCAAAGGCGGAACA 1137
Cebpd GTCCTGCTTTGCGCGTGTCG 1138
Cebpd GACGCCTTCAGACATAGCAA 1139
Cebpd GTTGCTGAACCTAACCTCGA 1140
Cebpe GTTTAGACCAAGTTGGCACT 1141
Cebpe GCAGCTACCAGCTTCTcCTT 1142
Cebpe GGTAGGTGGAGTTCAGGACT 1143
Cebpe GAAGCCTTCCCTAGCCCAGC 1144
Cebpe GGAAGCCTTCCCTAGCCCAG 1145
Cebpe GTAGATAGGGAAGCAGGAGA 1146
Cebpe GGGCTGCCAGGACATAGCTG 1147
Cebpe GATCCTTTCTGTTGGTTCTG 1148
Cebpe GAAACTGGTCCCGCTGGGCT 1149
Cebpe GGGTTAGTAGAAGATCAAGA 1150
Cebpg GAGCTATTCATATGAAGTAT 1151
Cebpg GAACTGTTCCCGGGAGACCC 1152
Cebpg GACTCCTGGGCATTGACTGC 1153
Cebpg GCCACCACCGACAGCCTAAG 1154
Cebpg GGATTCCTCGAAGTCTTATG 1155
Cebpg GCTTCTATTGGTCACGGCGG 1156
Cebpg GCATGATGCAGATCTGTGAA 1157
Cebpg GATAGAACTTTGCTTGCCAT 1158
Cebpg GGAGGACCACAGTGTGACTG 1159
Cebpg GAGGGTGTTCCTAGAATAGA 1160
Cebpz GTGACGCACTTCCTATTGCG 1161
Cebpz GTATGTCCAATGACCTATAT 1162
Cebpz GCTCCTCTGTGTACACACAC 1163
Cebpz GATTGCTTATTTGTGCCATG 1164
Cebpz GGTGGAACTTGGCCCTGGTC 1165
Cebpz GCCTTCCCTGTATTTGGAGA 1166
Cebpz GGCGGTGGCTCAACACCTGA 1167
Cebpz GTGTACACAGAGGAGCCCGA 1168
Cebpz GCCGCGCCATACGGTTTCCA 1169
Cebpz GAAGCTCACTCTCAGGGTGA 1170
Clock GAACCTAAGCGAGCAGCAGA 1171
Clock GCAGAAACTGTGCCTTTCGA 1172
Clock GGGTCGTCCAGGTCCATCTC 1173
Clock GGACAGAGTGGAGAATGGGT 1174
Clock GCACATGGTGTTTAAGGCCA 1175
C1ock GTGGTCCAGGCAGGACACTG 1176
Clock GACAATGAAACCATTAAAGG 1177
Clock GAGGACAATGAAACCATTAA 1178
Cnot3 GACTTAAGAAGGTGAAACCT 1179
Cnot3 GTACGTCGCTCTGCGCCGTT 1180
Cnot3 GAACTGCTTCTAGCTCTATC 1181
Cnot3 GAAGCTTATCTAGTGGGAGA 1182
Cnot3 GATCAGATAACAGCCTAGAC 1183
Cnot3 GAATTTCCATGGATCATTTC 1184
Cnot3 GCGTGGGACTGACGTTTCTC 1185
Cnot3 GTTTGCAACCTAGTCAGCAA 1186
Cnot3 GCATAGCGTGTGAGTGTTAA 1187
Cnot3 GACTGAGAAACACAAGGCGT 1188
Creb1 GAAACATGCTACAAGAAGAA 1189
Creb1 GCACGATCCGAGCCTCACTG 1190
Creb1 GCTAAGAACCGTGGGAGGAA 1191
Creb1 GCTATGGCACAGGTGGCATG 1192
Creb1 GAGCAGTTGCGGTAGCTTTG 1193
Creb1 GGCTCAGATGACTCCTGCAC 1194
Creb1 GGAACTTTGACGCGCCGCGA 1195
Creb1 GGTTTGTGTGTAGCCAGATT 1196
Creb3l2 GCCGGAGCTGGTTCTTTGCT 1197
Creb3l2 GAGCGTCGCAATGGACCAAT 1198
Creb3l2 GTCACTGGCCTGGAAGGAGG 1199
Creb3l2 GAAACATAGATCAATGAGCT 1200
Creb3l2 GGGCAGAGCTCAAGAGCCCA 1201
Creb3l2 GGTCAGGTCAATATAGAAGG 1202
Creb3l2 GAGGAGACTGAAGAAATCCA 1203
Creb312 GAGGGAACCCAGGTCACAGA 1204
Creb3l2 GAAGAGGCTAGTGTGGTCCA 1205
Creb3l2 GGGAGGGACATGGATGAGAA 1206
Crebbp GTGTGGCACACCCAAGTGAG 1207
Crebbp GGAAGTCCCTCTAACACTTT 1208
Crebbp GTTCAGAGGCCTCCGAATTG 1209
Crebbp GACCAGCATCACTGCATCTG 1210
Crebbp GCCATTACTAGCATAGGGCG 1211
Crebbp GTTGTCTACTAGTCTGTCCC 1212
Crebbp GTGAATGTAGGATGCTGGTG 1213
Crebbp GGGCCCATCTCAGATCCAGG 1214
Crebbp GTTTACCAACAGTATCCTTT 1215
Crem GTTAGCTACAGTACTACAGA 1216
Crem GAAAGAAAGATTGGAATTCA 1217
Crem GGACCAGACTCTCTTCAGGA 1218
Crem GCAAGTGAAGATTAAAGATG 1219
Crem GCAAATAGAACTTAGCATTG 1220
Crem GAACAACCATTTGTGAGTTT 1221
Crem GAAGGTTACAAATAGGCCAG 1222
Crem GCAGCCTCTTGGCTACTAAC 1223
Crem GACCTCAATCCCAAAGTGTG 1224
Crx GGTGTCACTGGGAAGCATGG 1225
Crx GTTCTGCTTCTCTAAACACC 1226
Crx GGGTGGTGGGATTAAGCAGA 1227
Crx GAAGGCTAAACTATGCAGAC 1228
Crx GAAACAATCCTTCAGGCCAG 1229
Crx GTCACTGGGAAGCATGGAGG 1230
Crx GATCTGGAAGGGTAATCCCA 1231
Crx GCTCTCTGAAGCTTGACAGG 1232
Crx GGCCCTAATCTCTCCTAGCA 1233
Crx GCCCTAATCTCTCCTAGCAG 1234
Crygf GGCAACAGAGGTGAATTGCC 1235
Crygf GTAGAGAGAAGAAACCTCCT 1236
Crygf GAAAGAATGGAAGGCAGGGA 1237
Crygf GGATAAGTCTGTCAGATTCA 1238
Crygf GAGATCATGATGAGTGTATG 1239
Crygf GCAGGAAGAGGTGGAAGGCA 1240
Crygf GAATCTGACAGACTTATCCC 1241
Ctbp1 GGTCTCTTTGGTTGGGTACA 1242
Ctbp1 GAAGGATGCTGAAGGCCATA 1243
Ctbp1 GTGTCCCAGAAGTTGAGGGA 1244
Ctbp1 GGAAAGTACAGCTTTGCCAG 1245
Ctbp1 GCAGGGACCATCCCTGGAGT 1246
Ctbp1 GTAGAGATGTGGAGATGCAC 1247
Ctbp1 GGTTTCCTGGGAGGCCCTAA 1248
Ctbp1 GCCTCAGCAGATATGTAGGT 1249
Ctbp1 GGTTTCCGAGGTTTCCTGGG 1250
Ctbp1 GGAATTTGGGCAGCCTGAGA 1251
Ctbp2 GTGAGAATAGAGGACCACGA 1252
Ctbp2 GGGATGTGATGTGTTGGACA 1253
Ctbp2 GGTCTTCAAAGTTGTGACCT 1254
Ctbp2 GCACACAGGACAGACCTTGC 1255
Ctbp2 GAGACACATTCATCTCCATG 1256
Ctbp2 GTGTCACACTCCTCCCTAAA 1257
Ctbp2 GAGTAGTGGGTTGGCCACCA 1258
Ctbp2 GCGCCTCCCTTGAGACTCTG 1259
Ctbp2 GAGCACAGCCACTGGAAAGG 1260
Ctbp2 GTGTGCTTCTAAGCCCAGGC 1261
Ctcf GAGTCACATTCCAAGGCTAT 1262
Ctcf GTCGGAGAAGTGAGAGAGTG 1263
Ctcf GGGATTAAGTACCACCGACT 1264
Ctcf GTAACCTTAGGACTGCTTTC 1265
Ctcf GGTATCAGAAGCCAGGAATA 1266
Ctcf GCAAATAAAGGCATTGTCTT 1267
Ctcf GATTAGAACACCTGCCAATA 1268
Ctcf GGGACAGAGTCACCTCAGTC 1269
Ctcf GGTGTGGTCTGCTATATCTC 1270
Ctcf GGCATTGTCTTTGGAAAGAA 1271
Ctnnb1 GTGAAGGAAGCGGGAGGTGA 1272
Ctnnb1 GAGTAAACTCTGCTGCTGGC 1273
Ctnnb1 GTTGATGACGTGTTTCTTTC 1274
Ctnnbl GTCTTCCTTCCCAGGGTTAT 1275
Ctnnb1 GGTCAGTAGAACCAGGCGTG 1276
Ctnnb1 GGAGGTGATGGGTACGGAGG 1277
Ctnnb1 GGATCCTATCCCAATAACCC 1278
Ctnnb1 GCTAGAGGAATATGAATACA 1279
Ctnnb1 GAAGCGGGAGGTGATGGGTA 1280
Ctnnbl GGTAACACACTTCACATAGA 1281
Cux1 GTGGCAGGGCTGCAAAGAAG 1282
Cux1 GACGCAATGTACGTCATATA 1283
Cux1 GGGTGGCAGGGCTGCAAAGA 1284
Cux1 GGCCATCTACGTTTGTGCGG 1285
Cux1 GTGCAATTGTGTCGTGGTAA 1286
Cux1 GAGGGCTCATATGATTACAA 1287
Cux1 GTCACCCTCCTTCCTGAGGG 1288
Cux1 GAAGTCTATGCAGCAAACCA 1289
Cux1 GTTGTCTTTGTGGGTGTCGA 1290
Cux1 GAACTCGCGCGCGCTAAAGA 1291
Cux2 GGTAAATATGCAGGCGACAA 1292
Cux2 GGATGCTTGCTGCGTTTCTA 1293
Cux2 GGACAATAGATCAATACCGT 1294
Cux2 GCAGGAATTTATTGCACCAC 1295
Cux2 GCCACTCGGAATTGCTAACT 1296
Cux2 GTCTTTCTGAGGCCCTGGGA 1297
Cux2 GCCTCTGTGGGACACACTGC 1298
Cux2 GAGATAGCGTCTGCTCCATC 1299
Cux2 GCGAATTTATGAGCCTTTAA 1300
Dbp GAAAGAAGTGGGCTTCGGGA 1301
Dbp GTGTTGGAGGGTCAGGTGAG 1302
Dbp GGCATATCCCTTCATCTCAT 1303
Dbp GGCGCAGTTCACTGAGTCGG 1304
Dbp GGCGGGCGTAATCCTCGTTG 1305
Dbp GTGAGGAAACTCAGAACAGG 1306
Dbp GCTGAGAATGGCCAGGCCGT 1307
Dbp GGTGTCAGTCACCTGGAGGG 1308
Dbp GGCCTTCTTCCCTCCCTACA 1309
Dbx1 GCGAAAGTGAGGGTTCGCGG 1310
Dbx1 GTGTACGTGCAAGATCTGTT 1311
Dbx1 GAGAAGTGTGCAGCCCTGCC 1312
Dbx1 GAACGCACTAAATTTATCTG 1313
Dbx1 GGACTCACTGTATAGCAGAG 1314
Dbx1 GGAGGGTAGCTAGCCTTCCA 1315
Dbx1 GTGGAATTCCCAGCCCGGTT 1316
Dbx1 GGAAGAACTAAGTTCACACA 1317
Dbx1 GACAGGTTTGCGCTAGCTAC 1318
Dbx1 GTGGCAAAGAGCGAAAGTGA 1319
Dbx2 GTTAACAGAAGGGAATAAAG 1320
Dbx2 GATCAGACAATTCTGTGCTG 1321
Dbx2 GGATGCTTCAAGACAAAGGA 1322
Dbx2 GGAGATAGGTGCACTGTGTC 1323
Dbx2 GAAAGGCAAAGTAAGGGTGG 1324
Dbx2 GCGACCAAGTACATGTACCC 1325
Dbx2 GATCTAGCTGAGAACCACAA 1326
Dbx2 GGACTCCAGCAGCAGGGTCA 1327
Dbx2 GTAACTATTGAGATGAGTGG 1328
Ddit3 GGATTGGCCACCAGTGGCCT 1329
Ddit3 GTTCAGGAAGGACAGCCGTT 1330
Ddit3 GCACAGCAGTGGCCAGACAC 1331
Ddit3 GTCAATCCAGGTGAACAAAT 1332
Ddit3 GGAGTCAGGAATGTCAGGTC 1333
Ddit3 GCAATTGCTTGGTGACCTGT 1334
Ddit3 GCCGTGAGACTCCTGAGTGG 1335
Ddit3 GAGAAGCGGGTGGACTATCA 1336
Ddit3 GACATGTTGACCTGGAGAGG 1337
Ddit3 GAACTCAGACAGCTAGAGGC 1338
Deaf1 GACAAAGGTAGACTATATGT 1339
Deaf1 GGTGTGATATGGTTGTATAC 1340
Deaf1 GGCTTCTAGAGCTGAAGTGG 1341
Deaf1 GTGTGCTCAGGATGAGCCAT 1342
Deaf1 GCTGAGAGCACCTGAGAGTG 1343
Deaf1 GATCACTGAGAGTCTAGGGT 1344
Deaf1 GAGTGTATTGTGGATATGCC 1345
Deaf1 GCATCTGAAGAGACCCAGGC 1346
Deaf1 GCAGGTGAGCACTTCAGCCA 1347
Deaf1 GTCTCCTCAGCAGCCAAGGA 1348
Dlx1 GTAGACCCATGGTCGCTCTC 1349
Dlx1 GTACAACAAATGGTCTAGTG 1350
Dlx1 GTCGGATGGCCGGATTGCCT 1351
Dlx1 GGGACAATTATTGCAGGTGA 1352
Dlx1 GACGCCTAACCCTGAACCGC 1353
Dlx1 GTTGAACCTACCTTCAGGGT 1354
Dlx1 GAGGAGGAGGTGGGAAGCTG 1355
Dlx1 GTGGTGTGTGGTAGTAGTGG 1356
Dlx1 GCTTCCCACCTCCTCCTCCA 1357
Dlx1 GCAATAATTGTCCCAGTGGT 1358
Dlx2 GATTCTGAGGTTCCCTCCTT 1359
Dlx2 GTAGGAGGTTGTTACAGGCC 1360
Dlx2 GCCTTCAAAGTCGTTTGCAT 1361
Dlx2 GTGGATCAAGCTACACTCTG 1362
Dlx2 GCATCCACTTCCCAGGCTAC 1363
Dlx2 GTCAGCCACTTTGCACCTGA 1364
Dlx2 GGAGCCTTATGTCCTGTTGC 1365
Dlx2 GAGATGTAAATCGTTAGACT 1366
Dlx2 GCCTTCAGGACAGGCTTGAT 1367
Dlx2 GGATGGACTCAGCGCAGTGA 1368
Dlx3 GCTGGCTTTCTGTGTTCTTC 1369
Dlx3 GTGTCTCTGTATGTAGTGTG 1370
Dlx3 GCTGAGGCACAGTTGATGGA 1371
Dlx3 GTTGATGGAAGGCCTGAAGC 1372
Dlx3 GGCTGCAAGTCTTGCCTTCG 1373
Dlx3 GGAGAAGCCTCCTTCCTCCA 1374
Dlx3 GCTCCCAAACCTATCCTTGG 1375
Dlx3 GTGGCTCTTCCATTCATGAA 1376
Dlx3 GGGCTTAGGTGAGATGAGGA 1377
Dlx3 GGTAAGCAGGCAGACAGGAA 1378
Dlx4 GCTGGAGGGAATCTGCTGTC 1379
Dlx4 GTAACGATGTTCAAGGTGCT 1380
Dlx4 GATGTGCTTTGAGGCAGGGC 1381
Dlx4 GTGATCCTGGAGCTCAGATT 1382
Dlx4 GACAGGTCCAACTTTCTTTC 1383
Dlx4 GCAACAGATGCTTGCATACA 1384
Dlx4 GAACAGAGACAGGCAAATCC 1385
Dlx4 GAATCTAGTTTGATGGCTCC 1386
Dlx4 GGAGATCCTCTTTGTCTGGT 1387
Dlx4 GATTCCCTCCAGCAGCCTCA 1388
Dlx5 GTTTCCAGTATCAGGGTCAT 1389
Dlx5 GCAAGGAACCAAGTCCGCTT 1390
Dlx5 GGCAAGGAACCAAGTCCGCT 1391
Dlx5 GGCCAGTCTTTCAGCACTTC 1392
Dlx5 GCTCCCTGCTGAGACATGTA 1393
Dlx5 GAGATTGGTGAATTTCAAAG 1394
Dlx5 GGAGAACAGCATTGTCTTAG 1395
Dlx5 GCAGCTCCAGATTCCAGAGA 1396
Dlx5 GCAGGAGGTCAGTCCCTCTC 1397
Dlx5 GAATCTTCTGGTTCCTCTTC 1398
Dlx6 GACTGGGTGGGAGAAATCTG 1399
Dlx6 GGTGTGTCTGGAGGTTGCGG 1400
Dlx6 GGTAAGCTCTAGGAGCTTGC 1401
Dlx6 GGTTCTCCTACCTGGTGGCT 1402
Dlx6 GTCCATCTTTGAAACAGAAG 1403
Dlx6 GCCTGTAATGATTATGGACT 1404
Dlx6 GCTCCCTTGGGAGTAGAGTT 1405
Dlx6 GAGTTACTGAACCGGCACCC 1406
Dlx6 GTCGAATGGTTTGTCTCCAA 1407
Dmbx1 GGAGCATGCATATGCAATTA 1408
Dmbx1 GATGAGCATAGGACCCAACC 1409
Dmbxl GACTGAACGGATGGAGGTCT 1410
Dmbx1 GTGTGTGTTCTATGCTTGTG 1411
Dmbx1 GCACACACCTCAGACACACA 1412
Dmbx1 GGAAGAGGTCGTTATGCAGG 1413
Dmbx1 GGGAAATGATGGACGCTGCC 1414
Dmbx1 GTAGCCAATCTTGCACTACA 1415
Dmbx1 GGGATCCTGGTGGGAGAGAA 1416
Dmbx1 GGCTCCCTGCCTCTAACTCT 1417
Dmrt1 GATAACAGATATTAGCTGCC 1418
Dmrt1 GAACCTTCCGAGGATTGCGT 1419
Dmrt1 GTACTGGTCCAAGCTGGAAG 1420
Dmrt1 GCCTCTTGGCTAACAGAGAC 1421
Dmrt1 GACACTGGCAGAGAGCAGGT 1422
Dmrt1 GTGGTCCTGAGATGGAAGCC 1423
Dmrt1 GAGGAGGCAGTGGTACACAT 1424
Dmrt1 GAGCGCCAATGGTTGCTTGG 1425
Dmrt1 GCAATTACATGTGTACCATC 1426
Dmrt1 GGTAGGTGAATGGTTGCATG 1427
Dmrt2 GTTCTCGAGAAGGTAACTAA 1428
Dmrt2 GGTGGTGGATAATACTAGGA 1429
Dmrt2 GTGTATGAACCAGTCAGATG 1430
Dmrt2 GCAGAGAGTAGAGCCGGGAG 1431
Dmrt2 GATAGGGAGCCCTAAGACAG 1432
Dmrt2 GAACTTAAACGCACCCACCC 1433
Dmrt2 GGCAAAGACCAGGCTCTCTA 1434
Dmrt2 GATCATGTGGATAACGGGCT 1435
Dmrt2 GACCACAAATGAGGAAACTA 1436
Dmrt2 GTGGGAAAGTGGTTCCCTGG 1437
Dmrt3 GAGGAGTTGATAGTTGTTCC 1438
Dmrt3 GTTACAATAGACTTTGAGGC 1439
Dmrt3 GATGTGCACTGGAGTGAAAC 1440
Dmrt3 GGGTGAAAGTTAACGTAAAC 1441
Dmrt3 GGGAATTGAGGGTACTCCGC 1442
Dmrt3 GAATGGCTGAGGCCAAGGGT 1443
Dmrt3 GCCAAGGGTGGGAAGGAAAG 1444
Dmrt3 GCTTTAACAACTCAGTGGGA 1445
Dmrt3 GAAGGGACCAGGGAAGGAAG 1446
Dmrt3 GAAGGAGCCAACGGAAGTCC 1447
Dmrta1 GTGCAGACTTCATCTAGGAA 1448
Dmrta1 GCGGTTTCTTGCTCTGGGAC 1449
Dmrta1 GCTCTCTGTTTCTACTAAGT 1450
Dmrta1 GGGCGGAGAGTGGGACTTTC 1451
Dmrta1 GTCTAGACTCAGAGGCTCAC 1452
Dmrta1 GACAGGTTAATTCAGAGTCA 1453
Dmrta1 GAGCACATGCAGATTATACA 1454
Dmrta1 GAGGACCTAGGGCGGAGAGT 1455
Dmrta2 GCTCCGAGGTAGTTGAGAGC 1456
Dmrta2 GCAGAAGCTAACATCAGGAA 1457
Dmrta2 GAGTGTGCATACTCGCGACC 1458
Dmrta2 GACTGTGTCACCCTCCATGC 1459
Dmrta2 GAAAGGCAAGGAGGGCACAG 1460
Dmrta2 GGCATTCACGTGAAGAATTA 1461
Dmrta2 GCTTGGACCCACGTTCCTCC 1462
Dmrta2 GCATTAAAGGTGATAGAGGG 1463
Dmrta2 GGAAAGGCAAGGAGGGCACA 1464
Dmrta2 GGGAGCACATATCCAACAGG 1465
Dmrtb1 GTCAGGGATGAAAGATTCGC 1466
Dmrtb1 GCCTCCTGACTGGAGAGTCT 1467
Dmrtb1 GCCCTGCTGTGAAATCTTTC 1468
Dmrtb1 GGAATAAAGGCCATCCTGGA 1469
Dmrtb1 GGGTGTCATCTGAAGTGGGT 1470
Dmrtb1 GGTGTCATCTGAAGTGGGTA 1471
DMrtb1 GCAAGTGAAGCAGGAATGAG 1472
Dmrtb1 GACAAAGCATGTGTTCCAGT 1473
Dmrtb1 GAAATCTTTCTGGTGATGCC 1474
Dmrtc2 GTCTGTATCTACTCTCTCCC 1475
Dmrtc2 GCAATCAGTGAGCTGGAAAG 1476
Dmrtc2 GATGTCTCCTCATGTATTGG 1477
Dmrtc2 GAGTGATGAGAGGTGTCCTT 1478
Dmrtc2 GGTGCTATAAGGCCACACAT 1479
Dmrtc2 GATTGTTGCCGCGGAGAAGC 1480
Dmrtc2 GCAAGATAATTGCATTTCCC 1481
Dmrtc2 GGATCAGCACCATGGCCAGG 1482
Dmrtc2 GGTGCTTTCTGCCCAGCCTG 1483
Dmrtc2 GAAGTGAACGCTTAAGCGGT 1484
Drd1a GATCACCAGTCTGTGGAACT 1485
Drd1a GCTCCAGCCTTGGCACACAG 1486
Drd1a GGACTGACTGAGTCCATATC 1487
Drd1a GGTGACCTGAGGGCAATTTG 1488
Drd1a GTGGCAGCAAGACTGCCAGT 1489
Drd1a GCCAGAATCTGGACGGTGAG 1490
Drd1a GAGGCTGCTGAGTTTATGCC 1491
Drd1a GGAGCACTTTCCCTCCCTGA 1492
Drd1a GCAACAATGTAGTAACACTT 1493
Drd1a GAATCTGGACGGTGAGAGGC 1494
E2f1 GCAATCAGAAATGCTGATGG 1495
E2f1 GATCAACACATTATCTGGGA 1496
E2f1 GGGAGCCAGGAAATGAGTAA 1497
E2f1 GTTAAGAATTGGAGAGGCCA 1498
E2f1 GAGTAATGTGGTCAGAGTTG 1499
E2f1 GAGCATTGGTTGCGGCGTGC 1500
E2f1 GGCCGTCTCCAGTTCTCATG 1501
E2f1 GCTACAGGGAGCTCTCAAGC 1502
E2f1 GCTGCTTCTCAGGCCCTTTC 1503
E2f2 GCGAATCTGTGAATGACCCG 1504
E2f2 GATTCAGGAAGGAAGAGTGC 1505
E2f2 GGTAAGACCAGGGAGTCGGA 1506
E2f2 GACAGGCACAGCGTGGGTGA 1507
E2f2 GTAAGACCAGGGAGTCGGAG 1508
E2f2 GGAATGGAGGTGGCAGGGAG 1509
E2f2 GGACCCTTCCATGGATTCCG 1510
E2f2 GGAGTTTCGCTGCCTGGGAA 1511
E2f2 GGAGTCACAGAGAAATCTCA 1512
E2f2 GAGAAAGCTGCTACTCGGCC 1513
E2f3 GGGATACGGTTTACGCGCCA 1514
E2f3 GGTAAGCAGGACAIAAACCT 1515
E2f3 GCTCTATGCAAATAGAGCCC 1516
E2f3 GCTTTCCTGCGGACGTTGGG 1517
E2f3 GGGCTAATCATGAAGCTGCC 1518
E2f3 GTCTGGAGAGAGGAGGGTCC 1519
E2f3 GGCAAAGTCCTACTCTCCCA 1520
E2f3 GGTTTGCAAAGACTGGAATC 1521
E2f3 GAGCAGGCTTCTTAGGAGGT 1522
E2f4 GCTGAGGCTCTACCACATAG 1523
E2f4 GTTAGACTGGGCTGGAGGGC 1524
E2f4 GCGCCATTTCCTGTTGGGTG 1525
E2f4 GGGCGTTACAGAGCAGGAAA 1526
E2f4 GGTTCTCGCTTCTCAACTGC 1527
E2f4 GGCTACAAGCAGGTGAGTGG 1528
E2f4 GCACTAGGAAAGGGATTACA 1529
E2f4 GTCAGTGGTGCAGTCCTACC 1530
E2f4 GAGCCTCGTTGGCTGGGCTT 1531
E2f4 GTCTCGGACCTCACAAACCC 1532
E2f5 GGCAGGTAAGGAAAGAGCTG 1533
E2f5 GCCTAGTAACGCACTCTCCG 1534
E2f5 GTCTACTTCCTTCACCGTCA 1535
E2f5 GCAGGTAAGGAAAGAGCTGG 1536
E2f5 GTAACGCACTCTCCGCGGAG 1537
E2f5 GAATGCCCAAATTAACAGTA 1538
E2f5 GATCAGGTGCAAGTATTGTA 1539
E2f5 GTAGAAGTAGAATACAACTG 1540
E2f5 GGACTTAGTGAGGGCGGAAG 1541
E2f5 GTCATACATCTTCATCAACC 1542
E2f6 GTGTGTGGTGGGATGGGTTG 1543
E2f6 GTTTGGCATTCAACAGAGGA 1544
E2f6 GAGAGTTTCTCAGAGCAACT 1545
E2f6 GACCTGGGACTTAGTGAGGG 1546
E2f6 GCGCTGCGCATGTGCAAACG 1547
E2f6 GAAGCTGCGGGAGTGAGACC 1548
E2f7 GTGAACCCTGGTTAGCACCT 1549
E2f7 GGACTTTGTTGCTTTAATTT 1550
E2f7 GGAACAGTCAAGAATATCTC 1551
E2f7 GTAATACACTCTGAAACCCA 1552
E2f7 GTTTCTAGTAAGGACTAGCT 1553
E2f7 GTGCTTTGTACTTACATAAG 1554
E2f7 GCCAGGTGACACGTGAACCC 1555
E2f7 GTCTTAGCCGTTCCGTGCAA 1556
E4f1 GTGGAGTTGACCTGAGCAAG 1557
E4f1 GGGCGTGGCTTGTGTTAAAT 1558
E4f1 GTTGCAATGTCAGAATTTCC 1559
E4f1 GCGAGCAGGGACTGAGCAAG 1560
E4f1 GCCAGACATCAGGGCGGAAG 1561
E4f1 GGAGTTGACCTGAGCAAGTG 1562
E4f1 GGTCCAAAGTGAACTATCCG 1563
E4f1 GCGGTCTAGCGCGTCAGTAG 1564
Ebf1 GATGACGTTATGCAAAGAAG 1565
Ebf1 GAAGAGCTGGACACCTGGGA 1566
Ebf1 GAAGCCCTAGCTTAAGACTT 1567
Ebf1 GGCTGCCAAGGACTCCTTGG 1568
Ebf1 GCGGTCTACTAAAGTCGTAT 1569
Ebf1 GTAGACAGATACACCGGAGG 1570
Ebf1 GGGCAGAGGGAAGGAGATGG 1571
Ebf1 GCCCAACAGCATTCGTGTCT 1572
Ebf1 GGTCTGTCCAGGGAGGAAAG 1573
Ebf1 GCTAAGGAGGAAATGAGTGG 1574
Ebf2 GTTTGTCAAGGTCTTAGGGA 1575
Ebf2 GTATGAGAGAAGCCGAGGAT 1576
Ebf2 GAGCTGATCAAAGTCTCCTT 1577
Ebf2 GATAACTGCCGAATGCAACT 1578
Ebf2 GAAGCAATCATTTCGTGCGA 1579
Ebf2 GCGGATTTGCCTCTAGATGC 1580
Ebf2 GAACTTGTCACTGGGAAGGA 1581
Ebf2 GACCCTACAGATTCATTCCC 1582
Ebf2 GGTTATTCTCACGTAGCTGG 1583
Ebf2 GCAGCTGATTGTCTGCTCCA 1584
Ebf3 GACCTCTCCTAAAGGTCAGA 1585
Ebf3 GCAGAGATGAAGTTGGGAAA 1586
Ebf3 GGGTGGAGACCCTTCCTGGA 1587
Ebf3 GGCTCCTCTGCAGCAGGCTA 1588
E3f3 GCTCACACTGGGTGAGCGAC 1589
Ebf3 GGCAAAGCCTGCTGAATACA 1590
Ebf3 GAGGAGATTCCAGGAGAGGG 1591
Ebf3 GGAATACTTCCCACCCTCCA 1592
Ebf3 GGAGGAACCTGTCTCCGACG 1593
Ebf3 GGGAGTGTGGATCCCTAGAA 1594
Egr1 GAGAGATCCCGCTGGTCTCC 1595
Egr1 GGTTGAGGATCCCACCTTTG 1596
Egr1 GGAGACTGGGCAAAGTCAAG 1597
Egr1 GAGAGCCTTAGACGCAGTGA 1598
Egr1 GCAAAGAGCCCAGGAGGGAC 1599
Egr1 GTGGGAAGGGTCTGTAGGTA 1600
Egr1 GGGAGGGCTTCACGTCACTC 1601
Egr1 GCCCTCCCATCCAAGAGTGG 1602
Egr1 GGATCTGTTGGTTCTTGTGA 1603
Egr1 GTCACTTTCCAGGTGTCACC 1604
Egr2 GGGCGTTTGAAGTAATGGCG 1605
Egr2 GAAGCTCTAAGCAAGGGCGT 1606
Egr2 GGTGTGTAGTGTGTAGCGTA 1607
Egr2 GATGAAGGCAGTGTCTTCCT 1608
Egr2 GAAGTGGTTCCATACCATCA 1609
Egr2 GTAGCGTAAGGTGTGTTGAG 1610
Egr2 GCTCCGGGATCTACGTAGCC 1611
Egr2 GCAAATAGAGGTCCCGGCGG 1612
Egr2 GTAACCTGAGTCCCACCGCC 1613
Egr2 GGCTCGGAGTATTTATGGGC 1614
Egr3 GCTACGTCACGGAGCTTTCC 1615
Egr3 GTTTGGAGGAGAACATTGGG 1616
Egr3 GAGTGGGAGTGTTGACAAGA 1617
Egr3 GTTGTCCTCATTGCTGCCTG 1618
Egr3 GGCTCAGATAAATAGACTGG 1619
Egr3 GGCTGGAGAGCCAGGCAATT 1620
Egr3 GCAAAGAGGGTAATCCTCTC 1621
Egr3 GGCACCCTCAGGCAGCAATG 1622
Egr3 GTGTTGACAAGAAGGAAGAG 1623
Egr3 GAATCACACCGGGTTGGCGG 1624
Elf1 GAGTAACATAATTAGATGGC 1625
Elf1 GTGGACCCAATTATTCTGCT 1626
Elf1 GGCTCAAGGCTTTCAGCATA 1627
Elf1 GCCATATATCCCTTCATATA 1628
Elf1 GGTCAAACATGCAAATGCAC 1629
Elf1 GACATCAGIGAGCGGGATCG 1630
Elf1 GGATGGCTGACTGAGCACTG 1631
Elf1 GCAAGAAGTCCACTGTTCAC 1632
Elf1 GGGTTAATGAGTAGCCAGGT 1633
Elf1 GCAGCTTGTTCCAAGGTGTA 1634
Elf2 GATTAAGCTACATATCCTTG 1635
Elf2 GGTGAAGGAGCGCGTGTGTG 1636
Elf2 GAGGATCGTTTATTAGCCAT 1637
Elf2 GACAGTAATATAACGCGATA 1638
Elf2 GAGGTAAGGTTAGGATTACT 1639
Elf2 GTCCCTGGAGGTCTTGGGAG 1640
Elf2 GTTGGGCGCTGAGAAGAGGG 1641
Elf2 GCTGCAAACGCAGGACATCC 1642
Elf2 GGTCCCTGGAGGTCTTGGGA 1643
Elf2 GGGAGTATAAATAGCCGGCC 1644
Elf3 GCAGCCCTGACCTAGAGGAA 1645
Elf3 GCAGATACTAATGGAGTGGG 1646
Elf3 GCAGGCAGATACTAATGGAG 1647
Elf3 GACGTACGCCGAAGACCTGG 1648
Elf3 GCTTCAGCAACCATCGCGTT 1649
Elf3 GAGTCATTACAAAGACAAAC 1650
Elf3 GGACGGAATCAATACTCAGG 1651
Elf3 GCTGGTTCTCCCACATTCCA 1652
Elf3 GAGAGCGCCACAGGCACCAA 1653
Elf5 GGAAAGCTTCACTATGCCTG 1654
Elf5 GCAAATCTCTAGCCATGGGT 1655
Elf5 GACGGCCTAGGCAGTCATCT 1656
Elf5 GAGGCCTTACTCAGGCTGCC 1657
Elf5 GAAAGCTTCACTATGCCTGT 1658
Elf5 GGAAAGGCCTAGGCTGGGTA 1659
Elf5 GTGTAGGCAGAGCAGAGGGC 1660
Elf5 GGTGTAGCAGGGTCCTGGAA 1661
Elf5 GGAACGGAACCCACGAAAGG 1662
Elf5 GCCTGAGATTGAGAGAGGAA 1663
Elk1 GTAGGACTCAACTCTGTGGA 1664
Elk1 GTGCTTTAATATTGGAGGCT 1665
Elk1 GAAACAGGACTTATTTAGAA 1666
Elk1 GCCAAGGATCCTAAGCACAG 1667
Elk1 GTGTACAGCACCACCTACTT 1668
Elk1 GCGTCCTCCTGCTTGCTGAT 1669
Elk1 GCAGTCCTCCTTGACCCAAT 1670
Eik1 GCTGGGAAGATGCAGTCAAT 1671
Elk1 GACAGGAGAAAGCCAAAGAA 1672
Elk1 GGACAACGTATACTGAACCG 1673
Elk3 GAGTTTAGGGACAGGAGGGA 1674
Elk3 GATCCTGGCCATTGTCCTCA 1675
Elk3 GTACCCTGTGGTTTCAAGAC 1676
Elk3 GAAGAGGCTTAAGTTATTTG 1677
Elk3 GTCGGATAGAGTTACTGTCG 1678
Elk3 GAGAGTTGGGCATTGCTCGG 1679
Elk3 GGCTGGAGGAACTGTATACA 1680
Elk3 GCGTACTTATCCCAGACCAA 1681
Elk3 GACACAAGGCTCCTAGTTTG 1682
Elk4 GTTGTCATCTTCTCTTTAAC 1683
Elk4 GATGGTACAAGGTAGACACT 1684
Elk4 GAAACAAGTCACACTTGGTC 1685
Elk4 GTGCACGGGACGGACTAACA 1686
Elk4 GGACCAAGCTAAGTTGGTAA 1687
Elk4 GAAATCAACACCCAATTCCA 1688
Elk4 GGTAGACACTTGGTAAATAG 1689
Elk4 GGACATTCGTACTTCCTCGC 1690
Elk4 GGCTTAGTTATCTTATGCTA 1691
Emx1 GGAGCCTGAGGATGACCTGT 1692
Emx1 GCAGAGATCCGGAGAAGGCA 1693
Emx1 GGTCTCCTTGGAGCAAGGTC 1694
Emx1 GGAGTGGCATCCTAGCTTCT 1695
Emx1 GTTCTCTGGAGAATCTAGGC 1696
Emx1 GTCGCATATGGCGGGAGAGG 1697
Emx1 GATGCAGAGTGGAGGGTAGG 1698
Emx1 GAGAGCCCTAACACCGAGTT 1699
Emx1 GCTTCTCCAGACCAAGGCTC 1700
Emx1 GCAGAGTGGAGGGTAGGAGG 1701
Emx2 GTCTCCTGTTTGGTTTCTTG 1702
Emx2 GCTAATGATGCTAATGCTGG 1703
Emx2 GGCCTCCAGTCTCTTGCATG 1704
Emx2 GAAGCGGGTTAGCCCTTGCC 1705
Emx2 GCTAGGCCATCTATGAGCTC 1706
Emx2 GGTGTGGGTGCAGTAGGAGG 1707
Emx2 GACATCTGTTGTCCCAGGGC 1708
Emx2 GAAGACTGGAGCCCAAAGAA 1709
Emx2 GACTGCAAACGCGTGGACCC 1710
Emx2 GGAAAGGAGTCTTGGGTCCT 1711
En1 GACTTTGCGGATAAATAATC 1712
En1 GTTCTGCCAGGATCTCCAAC 1713
En1 GTGGGTGAGAAGCTACAGCG 1714
En1 GAGAATCTCCCGACTTCTCT 1715
En1 GTGAGGGCAACTGGAGATTT 1716
En1 GCCAGGATGGCAGACAGGTA 1717
En1 GATCCGAGAAAGCTAGAATT 1718
En1 GTCAGAAACTATGACATTTG 1719
En1 GCTTGCCAGGACGTCAGCAC 1720
En1 GTGGAGAAGCCTCAGAAAGT 1721
En2 GTGCAGGAGACGCATGCATA 1722
En2 GAGGCACGTGTCCAGGAGAC 1723
En2 GAACTGCCAGGTCCTGGTGA 1724
En2 GAGCCTACAGAACCCAGGCA 1725
En2 GTGGCCTGGTGGCTCAACAT 1726
En2 GCAAGGGCAATAACTCCCAA 1727
En2 GGGCACGGCCACTTTAAAGG 1728
En2 GTGGCTCAACATAGGAAATG 1729
En2 GCCTCCTATAAGGAACTGCC 1730
En2 GGCCTGGTATGTAAGTGGGA 1731
Eomes GATGATACCATCTTGGCCTG 1732
Eomes GTGTTTCTTTAAGCGTCTTT 1733
Eomes GCTTGGAAACTTGTGAGCGG 1734
Eomes GGTGTTTCTTTAAGCGTCTT 1735
Eomes GACTGTTTGCGGAAACGCAG 1736
Eomes GGCACCGTTCAGACCCACTC 1737
Eomes GCCCGAGACCAAATCGGAGC 1738
Eomes GAGGGTGTGCGCAGAGACTT 1739
Eomes GCTCTATGGCGCCGGAGAAA 1740
Eomes GTCCTGCTGTTTGTGCACCC 1741
Ep300 GCTCCTAAGTCTAGTGTGTA 1742
Ep300 GTTTGGGATCCTCAAATATA 1743
Ep300 GGTACCTGGCTGGAGAGCAG 1744
Ep300 GAGTGAGGAGGGTACCTGGC 1745
Ep300 GTTCCAAAGATCAACCTGAG 1746
Ep300 GAACCTGCCTGAAACTTCCA 1747
Ep300 GCCGCTACCGCTATCCTGTA 1748
Ep300 GCTACCGCTATCCTGTAAGG 1749
Ep300 GAATCCTCCTTACAGGATAG 1750
Epas1 GAAAGCACGGTCCCTCAAAT 1751
Epas1 GACTTGCATAGAGCAGAGCC 1752
Epas1 GGGAGCCCACGGTGATACTG 1753
Epas1 GTTAGCGCAGGACTGAGTAA 1754
Epas1 GAAATCAGTTGACACACCTG 1755
Epas1 GAATAAAGATGGTACGGTTT 1756
Epas1 GAGCGCAGCTCCAGAGAAAG 1757
Epas1 GAGGATTGTACGGCCGCCTC 1758
Epas1 GACAAGAACAAGAGCCGACA 1759
Epas1 GGGCGATACCTGTAACCCGC 1760
Erf GAGAGTGGGTAGGAGAAGTA 1761
Erf GCTGAATAGGAACCCAACAA 1762
Erf GCTGCAGCAGATAGGAGGAA 1763
Erf GAGTTACCAAAGGAAGAGAT 1764
Erf GACCAAAGGCCCGAGCGTAG 1765
Erf GAGGAAAGGAATTATATGAA 1766
Erf GGAAGTGACCAGAATGCATT 1767
Erf GATGCGGCAAGCAAGAGGGA 1768
Erf GCGCACTCACACACGCTTGC 1769
Esr1 GTCACTGAGCATCTTATTCA 1770
Esr1 GACAGTAGTCAGTAGGCTAT 1771
Esr1 GTTTACAGACAGTAGTCAGT 1772
Esr1 GAGCGTGCAAACTATGGGTT 1773
Esr1 GCATCTGCTGTCTTGAGGTT 1774
Esr1 GTGGAAGTAAGAATGGTATC 1775
Esr1 GTTTGGTCCAGAGTCTGCAC 1776
Esr1 GACTCTACTCTTAGAGAAGC 1777
Esr1 GGAGAATGATGTTGGGTGTT 1778
Esr1 GAGTGAAGTGTTGGGTCGGG 1779
Esr2 GTGAGAGAGACAGGGAGACA 1780
Esr2 GCTGGGTTAAGCTTGCACTG 1781
Esr2 GGCAGGTAAAGGTGGTGTGA 1782
Esr2 GTTCACAGAACCCAAGGAGG 1783
Esr2 GAGTCCATCCTGGTGAGGAT 1784
Esr2 GACCTGGAAAGAGTGTGGGA 1785
Esr2 GCTCCCGGTTTGTGGTCACG 1786
Esr2 GCTTTCATAGACATCTTCCA 1787
Esr2 GGGACATTCTATCTCACAAA 1788
Esrra GGGTGGAGTGCTCACTGATG 1789
Esrra GCTCACTCTAACTAGTTATC 1790
Esrra GGTGGAGTGCTCACTGATGA 1791
Esrra GGAAGCCACATCGAACCTAC 1792
Esrra GTTCTGGATCTCAGCCGGGT 1793
Esrra GATGGGTGTGCCATAAGGGT 1794
Esrra GTGCGATGTGAAGAATGGAG 1795
Esrra GGGACACTGGTTTCAGCCCT 1796
Esrra GTAGGCACAGGCCGACTCAA 1797
Esrra GCGTCCTACTAGGAGGAACC 1798
Esrrb GTCAATTCAGAAGTCAACCT 1799
Esrrb GTATCTGTATCCCAGTAGAG 1800
Esrrb GCAGGAACCACAAGGCTATG 1801
Esrrb GTCATGTAGAAACCAACTCA 1802
Esrrb GTCCACCTCTTACATCATGG 1803
Esrrb GGTGAGTGAGTGACACCCTC 1804
Esrrb GCTTCAGGTATTGGAATGAA 1805
Esrrb GGTGGGACTGTTGGAAGGGA 1806
Esrrb GCAGGGAGACTGTGTAGGTA 1807
Esrrb GGTTAGTGGGCTCCAAGTGT 1808
Esrrg GAGAGGGCCTGTGCTTCTGT 1809
Esrrg GGGATATTAAGGCAGGATGC 1810
Esrrg GAAGAGGGTTGAAGGTAAAC 1811
Esrrg GACAAAGGTCTAAGGAGTAT 1812
Esrrg GAGCGATTGTAAATGTGTGA 1813
Esrrg GTGAATGCGTGCAATGAGCT 1814
Esrrg GGTAAGACTTCAAATGCAGG 1815
Esrrg GGGAGGGCGGGAAGTTGTTA 1816
Esrrg GCCAACTCACGAGCCAGGAA 1817
Esrrg GAAGACTTGCAGGAAGAGTG 1818
Esx1 GTGGGACTACACTGTAGGGT 1819
Esx1 GGAAACACTCCTATTTCTAA 1820
Esx1 GACATTTGAATTGGCTTCTT 1821
Esx1 GTAGTCCCACCCATTCCGAA 1822
Esx1 GGCATAAAGGGTTTCTTGCA 1823
Esx1 GAAGAAGCCACGGAAACCAA 1824
Esx1 GGAGTGTTTCCATTCGGAAT 1825
Esx1 GGGTGGGACTACATTGTAGG 1826
Esx1 GTCTTGCCCAACCATTCCAC 1827
Esx1 GTCTAGGCAGGAACCCTCGC 1828
Esx1 GCCAGATAACAAGATGAGTG 1829
Esx1 GAGCTGCCCTTTGTTTCTTA 1830
Esx1 GTGAGGAAATCCCTGTATAA 1831
Esx1 GACGGACTTTCCCGAGACTG 1832
Esx1 GAGTGTCAATCTCTGGGTCT 1833
Esx1 GCAAGAGTCACCTTTATACA 1834
Ets2 GCCAGGCTAGGCTTTAACTC 1835
Ets2 GGCACTTGGGTTGGGTGGTT 1836
Ets2 GTTGGGTGGTTAGGCTTCTG 1837
Ets2 GCTCAAAGGCTCTATCTTGG 1838
Ets2 GGCTGAGAACTTGGTAGGGA 1839
Ets2 GCCCTTTGAACCCAGAGGGT 1840
Ets2 GTGGGCCAACCACAAAGCAG 1841
Ets2 GTTCGGGCGTTATGCCCAGG 1842
Ets2 GCAGGGCTGAGAACTTGGTA 1843
Ets2 GGCAAGCTCAGGCAAGGCCA 1844
Etv1 GGAGCCGAAAGGTGGAGTGG 1845
Etv1 GGGTCAGCAATAAACAACAA 1846
Etv1 GCAGGATTTATTGAGATACT 1847
Etv1 GGACTTCTATCAACCTAGAG 1848
Etv1 GGAGTGTTAGGACATGCTCT 1849
Etv1 GAGAACGGGAGCCAAGAGAA 1850
Etv1 GAGAGGTGGCGCTGGAAGAG 1851
Etv1 GCCTTATCCGAATCACTCAA 1852
Etv1 GAGTCAAATAGTTAACAGGT 1853
Etv1 GAGCGAGAGATGCGAAGGGA 1854
Etv2 GGACAAGATGGTGACATTTA 1855
Etv2 GCATCAGCCTACGTCACAAT 1856
Etv2 GTTGAGAAAGGAAAGTTCTA 1857
Etv2 GGGTGACAGACAGCCAGATC 1858
Etv2 GCCTGGAGGATGAATGAATT 1859
Etv2 GGGTCAAGTTGCAGGGATGG 1860
Etv2 GTTCGTGGCTCACCTCTGGC 1861
Etv2 GTCTGAACTAGGAAGGACAG 1862
Etv2 GCTCTGGGCTTATCTGCAAC 1863
Etv2 GTGTCTGAACTAGGAAGGAC 1864
Etv4 GGTCAGATTCTGGGTCTCCC 1865
Etv4 GGAGGAACTCCGAGTCAGAC 1866
Etv4 GTGACAAGCTGAGTTACCTC 1867
Etv4 GAGGCGTGAGCTAACGCCAG 1868
Etv4 GCCATCTTACTCCTTATGAT 1869
Etv4 GGCTCAAACCGGCTTTCTCA 1870
Etv4 GAACCCGTGGAGAAGCTGCC 1871
Etv4 GGTCTCCATGAAGGTTCAGG 1872
Etv4 GAAAGCTAAGAAAGACACCA 1873
Etv4 GCCAGGGCTCTCCAGAGAAG 1874
Etv5 GCAGGACGAGGAGTTGGAAG 1875
Etv5 GTGTGAGTACGGGCTGCCCA 1876
Etv5 GGTCAGCGAGTTTCTGTGTG 1877
Etv5 GCAAGCAACACTGCTTCTCC 1878
Etv5 GATAGCCACAGTATCATATG 1879
Etv5 GGGATGAGAACAGGGAGGGA 1880
Etv5 GACACAAGAAGAATGTCCCA 1881
Etv5 GAGTCAGTGAAGCTCTTAAA 1882
Etv5 GTGTTGCTTGCCAAAGGATC 1883
Etv6 GAGTCTGGGAAACCCTCAGC 1884
Etv6 GAACCAGGCTTGCTGGTCCT 1885
Etv6 GGAGAAGGACATGTCAGGAA 1886
Etv6 GAGAGATGAACCAGGCTTGC 1887
Etv6 GGGAATACAGAGGTGAGTCT 1888
Etv6 GTTCTTGGAGGGAACCTCCA 1889
Etv6 GTCCAGTCACCTACGTCGGT 1890
Etv6 GACCTGGGCCACGCACAGTA 1891
Etv6 GGCATAGTGCATAGTGGCCC 1892
Etv6 GGAAAGCCACCCTGTGGTAT 1893
Evx1 GAGAGTGCTGGAGAAAGACA 1894
Evx1 GGCAGGTGGGCCAGATTGAG 1895
Evx1 GCGGCCAGTTCTTCGAGGAT 1896
Evx1 GGTAGGGAGAGGTTCAAGTA 1897
Evx1 GCATCGGCATAGGTAGGGAG 1898
Evx1 GAACAGAATTGTGAGATCAA 1899
Evx1 GCCCGGCTAGGAGGGATAGA 1900
Evx1 GCAGCTGTGGGTAGATTGTG 1901
Evx1 GAAGGTTATTTACTGAGCAG 1902
Evx1 GACCCAGGAAGGAGACTAAA 1903
Evx2 GAAATGCTATCCTCTGCTAA 1904
Evx2 GGGCGCGTCAAGAATGTAAG 1905
Evx2 GCTTGCCTGTAGAAATAAGT 1906
Evx2 GGCCTGCCTTTAAATAAGAC 1907
Evx2 GGTCTAGGCTAGGCTCCATG 1908
Evx2 GTGTCTCAAGGCGGGAAGGA 1909
Evx2 GCATCTGAGTCGGGCAGGGT 1910
Evx2 GTAAGGGCCTAGGGTGGAGG 1911
Evx2 GAAATCTTCCTAGGCCACTG 1912
Evx2 GCTTTCTTGCTACGTGGCTG 1913
Ezh2 GGTTCCTTTCGGCACCTTGG 1914
Ezh2 GATAACTGAACAGGGAGTGG 1915
Ezh2 GTTCGGCCCTCTGATTGGAC 1916
Ezh2 GTATGAATACTAGCTTCTAA 1917
Ezh2 GACACTGGTGGAAGTCATCC 1918
Ezh2 GGCGACCAGATTTCTCTGAA 1919
Ezh2 GAAAGCCATGGACAGGCAGG 1920
Ezh2 GCAGCTCATTTCTAITCCTC 1921
Fev GGATGATAGAGAAATTGTTG 1922
Fev GCTCAGTCTGACAGGGATCT 1923
Fev GAATCCTATGGAAACTGGGA 1924
Fev GCATATATTGGCTGTGAGAC 1925
Fev GCAGGGAGAAGAGTTCAGAG 1926
Fev GATGGCTAGAAAGAGGGCTC 1927
Fev GAGGGAGATGGCTAGAAAGA 1928
Fev GCCAGACGAGACAGGAAACC 1929
Fev GACTCTCA6CAAACATCGGT 1930
Fgf3 GTTCAACGGCTACATCCTGT 1931
Fgf3 GGATAGACCACTCCCACTTA 1932
Fgf3 GCAGAGACAGAAATAAAGGT 1933
Fgf3 GACTGCTTAAGATTTCTCAG 1934
Fgf3 GGATTCATTTGTGACATCTT 1935
Fgf3 GCATCCTTCATTTGAGTCCC 1936
Fgf3 GTCACAGAGCCTTAGAGCCC 1937
Fgf3 GGCAGAGACA6AAATAAAGG 1938
Fgf3 GAAGGACCACACCAGGGTGC 1939
Fgf3 GCCAGGCACAGGAAGGTAAC 1940
Figla GGAAATATTTGCATGCATTC 1941
Figla GGAACAAAGCCCGTAGACCA 1942
Figla GGGCCAAATAAATAATGGAA 1943
Figla GCTGCGACTTCTTACTTTCC 1944
Figla GGCTGAGGGTGTGACTGCTG 1945
Figla GTTGAGATCATTTCCTCACA 1946
Figla GGACTCTAGGACAGGAAGAG 1947
Figla GGCATCTGAAACCAGGAGGA 1948
Figla GCACGTGTGCAGCCTGAACA 1949
Figla GACTTAACCTGACTCACCTG 1950
Fli1 GTAAACCGAGTCTCAATTGC 1951
Fli1 GGAAAGAGGCCAGAGGCGTT 1952
Fli1 GCTGCCATTCCTGAGCTGCA 1953
Fli1 GGCGTTTGGCTTTGGATTTG 1954
Fli1 GGTGGTAACCACATTAAACA 1955
Fli1 GCTGGCCAGGAACAATGACG 1956
Fli1 GAGGGTCTCCTTCCAGGCAC 1957
Fli1 GAAGGGAAGAGCAAGAGGGC 1958
Fli1 GCTTAACCCTTTCCTGCCTG 1959
Fli1 GAGGGTGTGCACCACTGTGT 1960
Fos GGATGGACTTCCTACGTCAC 1961
Fos GATCTAAGGATGGAGTAGCA 1962
Fos GCAGTTATGAGTGGAAGGCA 1963
Fos GAGGGTTCAAGACAGGACTC 1964
Fos GAGAGGATTAGGACAGCGGA 1965
Fos GGCCGGTCCCTGTTGTTCTG 1966
Fos GAAAGATGTATGCCAAGACG 1967
Fos GATCCAAACCCAGCGGGAGC 1968
Fos GTAAAGGAO6GAGGGATTGA 1969
Fosb GGTGAGTCTTCAGGCTTTGA 1970
Fosb GAATCCGTGACAAAGCTAGT 1971
Fosb GTCACGGATTCTGTGTGACT 1972
Fosb GTCTCCTGAGCTAAGTGGGA 1973
Fosb GATATCTCCAGGTGTAGGGA 1974
Fosb GAGTTGCACCTTCTCCAACC 1975
Fosb GCGGGAAGGGAGAGTTTGGG 1976
Fosb GTATAAGCAGACCTGGGATC 1977
Fosl1 GTTCCCAATGAAGACAGCCC 1978
Fosl1 GGAGTGTGTGTACGTGAcTT 1979
Fosl1 GCTTTAATCCAGGCCTCTAC 1980
Fosl1 GCTCTCTGCCTGTAGAGGCC 1981
Fos11 GAGAGGAGCGGTCTTAAGTC 1982
Fosl1 GAGCATCACCTCCTGCTCCC 1983
Fosl1 GAGCGCCTACAGAAGGACAT 1984
Fosl1 GCTTGTGATAGCTCCAGAGA 1985
Fosl1 GCTGTCTTCATTGGGAACAA 1986
Fosl1 GTCCTGAGACACAGTCAGTA 1987
Fosl2 GGTAATCCCAAACAGTACTA 1988
Fosl2 GTACAGATAAGCGCTGTACC 1989
Fosl2 GGGCTGGAGAATAAAGAGTG 1990
Fosl2 GGAAACGCAGGCGCTTTATA 1991
Fosl2 GCGCCCTTGGTCTGTTCCAT 1992
Fosl2 GCCTGAGTTTCCCGGCGACT 1993
Fosl2 GGATACAGATGCACTGCATA 1994
Fosl2 GTACACGCACGCACCAGCCT 1995
Fosl2 GAGTCGCCGGGAAACTCAGG 1996
Foxa1 GCACGGAGTGTGTGTGTGTT 1997
Foxa1 GGAGTGACTTCTAGTCACAG 1998
Foxa1 GTACGTTCCCGCAATGCCGG 1999
roxa1 GCCCTGTCTTCTATGTCATA 2000
Foxa1 GCCTTCACTTTCTGCTTAGT 2001
Foxa1 GCTTAGTTGGTACCCAGATA 2002
Foxa1 GGGTCAAGAATCAGGATGAG 2003
Foxa1 GCAGGAACAAGGAAGCTTCT 2004
Foxa1 GCAGATGCGTTCCAGCACCC 2005
Foxa1 GATCAGTAGGAGAGCAGAGA 2006
Foxa2 GAAGTAGTGCTGGCGGCAAT 2007
Foxa2 GAAATAGTTGGCCCAAATCC 2008
Foxa2 GTTTAGCTGCAGCCAATACC 2009
Foxa2 GTGTGAGCTGATTATTCAAA 2010
Foxa2 GGCTGGTCACTGAATGCCAG 2011
Foxa2 GACCCATTTGAGTAGAAGGA 2012
Foxa2 GAATTGCACAGCGTTAAGCA 2013
Foxa2 GGAGCACTTGGGTGGAGATG 2014
Foxa2 GATTATTCAAATGGGCTGCC 2015
Foxa3 GTGTGGTCGAACTTGTTATT 2016
Foxa3 GATCCACTCTTTAGGATAAC 2017
Foxa3 GGTGGAGGAGGAGGAGGTGA 2018
Foxa3 GCTCCCGCTCTGTTGCTCTA 2019
Foxa3 GGAAGGAGGGAGGCAAACGG 2020
Foxa3 GCTCCGTACAGAGTCAGGGT 2021
Foxa3 GGAAACGTGCTGTTATCCTT 2022
Foxa3 GAAGCCAAAGAAGGCAAGGA 2023
Foxa3 GCGGATTTGAGGAAGGAGGG 2024
Foxa3 GCTTCGCACACGGCCAGTCT 2025
Foxb1 GATAGATATATTTGGACAGC 2026
Foxb1 GTATTTAACCTAGTGGCATG 2027
Foxb1 GTATCTTACTGGTTGCCATT 2028
Foxb1 GAAAGAAACCAGCGCTGGCC 2029
Foxb1 GAAGCATTGACCCGTCTCTG 2030
Foxb1 GATTGGATGGGTTGTTCAAA 2031
Foxb1 GCAATCGCGGCTTTAAGCCA 2032
Foxb1 GGTCCAACTGATTTAATCTT 2033
Foxb1 GAAGCTTGGTGGAGGTGGGA 2034
Foxb2 GGTCTGCTGTTCCACAGCAA 2035
Foxb2 GGTGTATCCTCTTGTTTCTT 2036
Foxb2 GGATGACTAGACTTGAGCTC 2037
Foxb2 GACCAGTGGAAATGGAGAAG 2038
Foxb2 GCCTCTCAGCTGTAAGGTTT 2039
Foxb2 GGGAAGTGAAAGCGAAGGGT 2040
Foxb2 GGCTGCAGCTGCAGCTCAGA 2041
Foxb2 GGAGCCAGAAGGTTCCCTGC 2042
Foxb2 GCCAAACAGCAGGAGCCAGA 2043
Foxb2 GGGCGTCCTAGGAATTCCTC 2044
Foxc1 GACGGCAAAGTGATTGCCCG 2045
Foxc1 GAGTCTGGGAGTGAGTGGGT 2046
Foxc1 GGAAAGGAGAGGACAAGGGA 2047
Foxc1 GTGGTGACACCACAGGAATG 2048
Foxc1 GAGGGATATTCGGAAAGGAG 2049
Foxc1 GCGGTATTGGAGGATCTGAG 2050
Foxc1 GAACGTGAAGATGCAGTCTT 2051
Foxc1 GTGTGTTAGTGAGGGAAAGA 2052
Foxc1 GTTTCCACATTCCAGCGGGC 2053
Foxc2 GAGTCGCTGTGCGTCAAGGT 2054
Foxc2 GAGGGAAGGAGCACGCTTGA 2055
Foxc2 GAGTCCTCCAAACAATTTCG 2056
Foxc2 GAAAGCGCTGTCTGGAGGTT 2057
Foxc2 GGTTTAAATTTGGCATACGC 2058
Foxc2 GGCTGGGAGGGAAGGCTTAG 2059
Foxc2 GTCCGGTGTAGATCTGGGTA 2060
Foxc2 GAGATCTGGCTAAGAGCATC 2061
Foxc2 GCTGGGAGGGAAGGCTTAGT 2062
Foxc2 GTGGGAATGCCAAACTGGGA 2063
Foxd1 GATCTAGTAGTCTCCTCTGA 2064
Foxd1 GAGAAAGTTCACCCGCAGGA 2065
Foxd1 GATCAATGAAGGTACAGAAC 2066
Foxd1 GATTCTGAGAGCTAGGGACC 2067
Foxd1 GGAGAAGAAGCCTGTTGTGC 2068
Foxd1 GCTTTCAGGCCAAGGAGTGG 2069
Foxd1 GGTAGGGTGTCCCAGCTCTC 2070
Foxd1 GCAGACAGGCGTCCTAACCA 2071
Foxd1 GGGCCTGTGACAAAGATGAA 2072
Foxd1 GAAACAGCCCTTTAACCCTT 2073
Foxd2 GGAGTGCACAGCAGGTATTG 2074
Foxd2 GGGATGAGGTTAAGTTTCTT 2075
Foxd2 GTGGCCAAGGCTCCAGCATA 2076
Foxd2 GCAACACTGGCCCAGGGATG 2077
Foxd2 GCGGTGCACACCAGGAAAGT 2078
Foxd2 GCCAGAACATTCCACTTCCA 2079
Foxd2 GCCCTATTCTCTGGGAGGGA 2080
Foxd2 GGGTGCCCTATTCTCTGGGA 2081
Foxd2 GCCTAAGGCGGAAGAACTGT 2082
Foxd2 GCCACTGTGAGGCGCTGTTG 2083
Foxd3 GACTTTGTCCGCCTCGTTGA 2084
Foxd3 GCTGGAAACGGAGCAGGCAT 2085
Foxd3 GTTATGACGTCTTTGTTTAT 2086
Foxd3 GGGAAATCCCAGAGATGCTG 2087
Foxd3 GGGCTCCAAGCAGCTCTGGA 2088
Foxd3 GTTCAGGGAATTGTCAACAA 2089
Foxd3 GCGGTCTTGGGTAAGTGGAG 2090
Foxd3 GGCTTAATATCGATTTCTAG 2091
Foxd3 GCTGACTAGACAGTCTTCTC 2092
Foxd4 GCTGGCCTCTGACCTCTACA 2093
Foxd4 GAACTTCCACAGTTTATGCT 2094
Foxd4 GTAGTTGGTAAAGACAACGA 2095
Foxd4 GGAACCGAGTCTCTCCAGCA 2096
Foxd4 GAGGGAAGGAGCCATTTCTC 2097
Foxd4 GCAGTGTGGATGCCTTACCA 2098
Foxd4 GAACCGAGTCTCTCCAGCAG 2099
Foge1 GCCAGTACCTTTCCTGAGCA 2100
Foxe1 GGGTAAGAACTGGACTAAAG 2101
Foxe1 GTCTACAGCTGAAGACGACG 2102
Foge1 GGTGGAAGGTACAACCCAAG 2103
Foxe1 GAATTCTGCTTCCCTCTGCT 2104
Foge1 GAAAGCCTCCTCGCCGCATC 2105
Foxe1 GAAGCAGAATTCTGGAAGAA 2106
Foxe1 GCCGCATCAGGGTCCTTAGG 2107
Foxe1 GTTTGCTGGCGCCTTTAAGG 2108
Foxe1 GGAAAGAGACACACTGTGGA 2109
Foxe3 GCTAGCAAAGACTGCTGGAG 2110
Foxe3 GAGCGGGAACTAGAAGCATG 2111
Doxe3 GCATCCTATGTAGCTGGTCA 2112
Foxe3 GGGATGGTACTTACTGAGAC 2113
Foxe3 GGGCTGGGAAAGCAAATTAG 2114
Foxe3 GGGAAAGCAAATTAGAGGGC 2115
Foxe3 GAGGAAAGCGAGAAAGGCTA 2116
Foxe3 GGACAGTTACACACAGGGAA 2117
Foxe3 GACTGACTCAGGGATGAGGG 2118
Foxe3 GACCCAGAGGGACTAGACCA 2119
Foxf1 GCGGATTTCAGAGTTAAGCG 2120
Foxf1 GCAAGCCTGCGCGTCTAAGT 2121
Foxf1 GGACAGACTTTAGAACTCTG 2122
Foxf1 GAGCCCACTGAATAGCTACG 2123
Foxf1 GGTGATTAGAGGATTCGCTT 2124
Foxf1 GAGCCACAGGATCACAAGAA 2125
roxf1 GGGTGTGGGAATGTGTGGCC 2126
Foxf1 GGCACATCTGTGCGAGGGTC 2127
Foxf1 GTCTGTCCTGGAGAAAGGAA 2128
Foxf1 GACCCTCGCACAGATGTGCC 2129
Foxf2 GGCACGGATCGCTAGGTTGG 2130
roxf2 GGGCACGGATCGCTAGGTTG 2131
Foxf2 GGGTCTGAGGAACAGAGGAA 2132
Foxf2 GGGATCAGCATGAAATAAAT 2133
Foxf2 GGAGCTTTGTGGGCAGACTT 2134
Foxf2 GGAGTAATCGAGTCTGGCCA 2135
Foxf2 GCTCAGAGAGAGATGGCCCT 2136
Foxf2 GCCTGGGAAGATGGGAGACA 2137
Foxf2 GTTGACAGATGGTGTCAGTT 2138
Foxg1 GAATGCGAGTCTCTCAAAGC 2139
Foxg1 GCTTTATGTGCAGAGGGAAG 2140
Foxg1 GCCCAGCATTTCCCAGGGAT 2141
Foxg1 GATTACCTTCAGAAGACAGA 2142
Foxg1 GTGAAATGATTCGGTGTAAC 2143
Foxg1 GTAGGAAGAGATCCAAGCAG 2144
Foxg1 GCGCGCCACGTTGTAAGCAG 2145
Foxg1 GCTAGAGAGATCTGTGAGCC 2146
Foxg1 GGTGCCAAAGGTTGATTTCT 2147
Foxg1 GCTCACAGATCTCTCTAGCT 2148
Foxh1 GTGAGGGTCGGGTCTATCTG 2149
Foxh1 GACTTTCCTGTGCTTCATGT 2150
Foxh1 GCTTTAACTGGAACCAAGGA 2151
Foxhl GTCATCCACTGTAGATTGAC 2152
Foxh1 GTCATGGTGATGGGACTTTC 2153
Foxh1 GAGGTTCCAGGIGAGAAGGC 2154
Foxh1 GGTACAGTCATGAGTGGAGG 2155
Foxh1 GCAGCAGTTTGGGTGATGGT 2156
Foxh1 GGAGTCTGCTCAGGACTTGA 2157
Foxh1 GATGGATTTGCCCGACCAAC 2158
Foxi1 GCTTACTTACTTTGAACCTC 2159
Foxi1 GGCAAATGAAAGCAATTCTG 2160
Foxi1 GAGCATGTGTCAGTGCCTGG 2161
Foxi1 GGATAAGCCACCTTTAAGCT 2162
Foxi1 GTGACCTGGCACAACTGTCC 2163
Foxi1 GAAGATGATGGCACCAAGAG 2164
Foxi1 GTAAAGCAGAGAGAGAGGTT 2165
Foxi1 GCCTAGCTCCCTCAGTGCCA 2166
Foxh1 GAAATCGTCCTTGCTGAGGG 2167
Foxh1 GACTCAGGAGAAGAAAGTAG 2168
Foxi2 GTTAACGAGGCAGTATGACA 2169
Foxi2 GTGCCTAAGGCAAGGGCATC 2170
Foxi2 GAGTTCCAAGACTGTTTGTC 2171
Foxi2 GACCTGTTTGTCATGTAGCC 2172
Foxi2 GGTTACAGCTGTGGCACAGA 2173
Foxi2 GCCCAGGCTACATGACAAAC 2174
Foxi2 GGTTGGTTAAGTAAAGGCAG 2175
Foxi2 GTCCAATCTGGCCTGGCTTC 2176
Foxi2 GAGAGAGAGGCTGGTGGCTT 2177
Foxi2 GAGAGCAGAGCCTTAGGAGC 2178
Foxi2 GGCCACTTCCCTGCAGTGCT 2179
Foxj1 GGGAAGCAGGGTGTTCAGAA 2180
Foxj1 GAATGAGCAAGGCAGAGCAA 2181
Foxj1 GAGACTTGGTCTGCAGAATC 2182
Foxj1 GGGCAAAGACTTCAAGGGCA 2183
FoKj1 GGCAGATGCAGAAGCAGGTA 2184
Foxj1 GGCAACTCTCTGGAACTCTC 2185
Foxj1 GGGAGGAATGCACTAGGGTA 2186
Foxj1 GCTTCCAACCAATAGTTCGG 2187
Foxj1 GACTTGGTCTGCAGAATCCG 2188
Foxj2 GCTGTCTGGAAGAGAAAGAG 2189
Foxj2 GTCAGTGAAAGATTGGATCG 2190
Foxj2 GTAGTGAGACCTGAAGGACC 2191
Foxj2 GATGCCAGCGTCCACGCTAA 2192
Foxj2 GGAAGGGTAGTGAGACCTGA 2193
Foxj2 GTCACTTGACTTTAGCACAA 2194
Foxj2 GGTCTCACTCCGGGTCCTTC 2195
Foxj2 GGGAGGGAAGAGGCTTTGTT 2196
Foxj2 GAGAAAGGGAGCCATGCCTG 2197
Foxj2 GGTCCTGGCTTGTGCGTATC 2198
Foxk1 GACACAGACCTTCCAGTGCT 2199
Foxk1 GATGAATCCAAGCACCCTTC 2200
Foxk1 GGTGGAGCAATTGAGCACAC 2201
Foxk1 GGGAATTAGCATCCCAGTGC 2202
Foxk1 GAGGCTGGAGTTTAAAGTCC 2203
Foxk1 GCACTGGAAGGTCTGTGTCT 2204
Foxk1 GCTGACGGCCAAGTGIGGGA 2205
Foxk1 GAGGGTGAGCTGGCACAGGT 2206
Foxl1 GAGGCAGGATGTGGAGGGAC 2207
Foxl1 GAAACACACTCCGACCCTCT 2208
Foxl1 GAACAGAGACTGCCTCCTCA 2209
Foxl1 GTTGAAGTCACAGAGGAAAG 2210
Foxl1 GAATCTACAGCGGAATGTGG 2211
Foxkl1 GGTTGGACACTTAAGGAATC 2212
Foxkl1 GCACCGCCCACTTTAGTCGT 2213
Foxkl1 GTGGGTGGGTGTGTGTGTGG 2214
Foxl2 GCAGAGCCTCTAACTTCTGC 2215
Foxl2 GACTTCTTGCTGTCCTTTGC 2216
Foxl2 GATGAGACCCAGGGTCAGCT 2217
Foxl2 GGTGGAGTGGCCGAACTTTG 2218
Foxl2 GAGAGGTGATCCAAGCCTCT 2219
Foxl2 GCATCTTCTCCTTCCAGCAT 2220
Foxl2 GCTTCCCACTTTGAGATGAA 2221
Foxl2 GCACAAGTGTCACGCGTGGA 2222
Foxl2 GAAGTCAGCCTCTGGCCATC 2223
Foxl2 GAAGGAGAAGATGCAGGTAA 2224
Foxm1 GTTGAGTGTGGAGAATAATG 2225
Foxm1 GCCTTCTAGTACACAATGGC 2226
Foxm1 GCCACGTAACCGCAAGTCTA 2227
Foxm1 GTATGAAGAGAGCTGCAGGG 2228
Foxm1 GCAAGICACTTGCAATGACT 2229
Foxm1 GCCAAGGCGTTGTCACAGAA 2230
Foxm1 GGGAAGAAGGTCTGAGCCTC 2231
Foxm1 GAAGAGAGCTGCAGGGTGGA 2232
Foxm1 GCAAGCTTTGACCCTGAGGA 2233
Foxn1 GACATGGGAGGGAAGTCACA 2234
Foxn1 GCAGGCATGCCCACAGACAT 2235
Foxn1 GTTAACCATCGTGTACAGAT 2236
Foxn1 GGGTTCTGGGAAGCAGCACA 2237
Foxn1 GAGGGAAGTCACATGGATTT 2238
Foxn1 GTGCATGTCCCAACAGGCCT 2239
Foxn1 GCTACACACTGCCACACATA 2240
Foxn1 GGAGACACAAGCCTGAGTAC 2241
Foxn1 GAGTCAATTCACCGTTCTCT 2242
Foxnl GGACATGCTGTGTATGGATG 2243
Foxn2 GGAGATTCTTGTATACCAAG 2244
Foxn2 GTATTGCCTACACTGTATTG 2245
Foxn2 GGTCTGGGTGTGGTCAGTCA 2246
Foxn2 GCTTCATTTGGTTCCTGATG 2247
Foxn2 GCTTTGTTGAGCAAGCAGAC 2248
Foxn2 GGTGTGGTCAGTCACGGCAG 2249
Foxn2 GCTCCACTTAGTTCAAAGTC 2250
Foxn2 GGCAACAGTGATGCTATGTA 2251
Foxn2 GACAAAGGTGCCCAGGCTTG 2252
Foxn2 GCCCGGAAGGACCTATGGGA 2253
Foxn4 GCCACGGTCGCATGTTGAAG 2254
Foxn4 GCAGTGCATGACCCAGCCAG 2255
Foxn4 GACCGGTTTACGTATTACTC 2256
roxn4 GAGTTGGACAGCTCCAGAGA 2257
Foxn4 GTGCAGTGCATGACCCAGCC 2258
Foxn4 GAAGCAACGGGCTCTTTCTG 2259
Foxc8 GCGAGCTCGGGAGACGAAAG 2260
Foxn4 GATCAATCCTGTTAGGGAAC 2261
Foxn4 GCCTGAACTTAAGGGTTCCC 2262
Foxo1 GGTTCAGGATGAGTGGAGGC 2263
Foxo1 GAAGACTTCACTCATCTTGG 2264
Foxo1 GAGGCGGCAGTAGGTTGGTG 2265
Foxo1 GCACCTTAAACGGTTCATAG 2266
Foxo1 GGTGAAGACCCGTCGCTCTG 2267
Foxo1 GTCCTCGGCACCTCTGGTTC 2268
Foxo1 GCAGGTGTGCACAGGTAGGG 2269
Foxo1 GACGTCACTGAGCATCTTAC 2270
Foxo1 GCGAAGGCCAAATTCACAGC 2271
Foxo3 GTCTGGAGCCCAGAGACTGG 2272
Foxo3 GGGAGGAGGGAAAGGAGGTA 2273
Foxo3 GTGCACACACCTGGACCACA 2274
Foxo3 GCGTCGAACTAGCTTGGTGC 2275
Foxo3 GGCAATATAGATGGTGATGT 2276
Foxo3 GCATTCTGACCCTGAAGGTA 2277
Foxo3 GAAGAGGAGCGAGAGGCGTC 2278
Foxo3 GAGGCACGGATCGTGGGATA 2279
Foxo3 GACAGCGGGAGGACTAGAGG 2280
Foxo4 GAAGTAGCAAGTTACAGAAG 2281
Foxo4 GGGATTCAGTTCTGGAGTTG 2282
Foxo4 GTTTCCTCTGTCAGCTATGC 2283
Foxo4 GTAGTCTTCGAGAACGACCA 2284
Foxo4 GGTGGAACTTTAATGATTAG 2285
Foxo4 GGTAAACAGAGACGTCTGGC 2286
Foxo4 GGTCACTCTTGAGAGGGTCA 2287
Foxo4 GTCTCTGTTTACCACTCGCT 2288
Foxo4 GAAGGCCCAGTGTATGAAGA 2289
Foxp1 GGGAAGGAATCACACCACCA 2290
Foxp1 GCAGTGAGGGTTTCTAACCG 2291
Foxp1 GGTGGCCTCTGGATCCGCAA 2292
Foxp1 GACAGTCTTCTGAAGCAGGC 2293
Foxp1 GTAATTTGTCTGTAGAACCC 2294
Foxp1 GCAATTAAGAATTCACTCCA 2295
Foxp1 GAAGGCTAGGAATCTTCTTC 2296
Foxp1 GCTTTGGTGTTGATGACAGT 2297
Foxp1 GTAGAGTAGTAGGGTCTCAG 2298
Foxp2 GAGCTGCTGGCAAATGAAAC 2299
Foxp2 GAAACTCTAACTGCTTGCTT 2300
Foxp2 GTAGAGAAGAATGACTACAG 2301
Foxp2 GACCACATACCTTGCCACGG 2302
Foxp2 GGGTCTTGTGACTTGAATCT 2303
Foxp2 GAGGACCCTGTCAGAATGAA 2304
Foxp2 GTAGCCTAGCAGGGTTGGTG 2305
Foxp2 GGCGCACACACAGGAGAGAA 2306
Foxp2 GACCCTGTCAGAATGAAAGG 2307
Foxp2 GAGAGAGGCGACTTGAGCAG 2308
Foxp3 GGGTCTGTGGAAGCTGAGAC 2309
Foxp3 GAGCAGGGACCATTAACTTT 2310
Foxp3 GCTAAGGAAATACTGAGGTT 2311
Foxp3 GAGAAGACAGACCCATGCTG 2312
Foxp3 GGGATGAGGTCCTCTTACTT 2313
Foxp3 GGCAGAGAGGTATTGAGGGT 2314
Foxp3 GCCATTGACGTCATGGCGGC 2315
Foxp3 GGCAACAAGGAGGAAGAGAA 2316
Foxp3 GTAGCCTTTCTTTCCACAGA 2317
Foxp3 GCCCAAGTGTACAGGGAGCA 2318
Foxp4 GGAGGACTAAATTGGGTAGC 2319
Foxp4 GGATGAACTGGGTAAGGACT 2320
Foxp4 GAGCTTGTGTTTAGCACTTC 2321
Foxp4 GGACCACGTGGACCAAACTT 2322
Foxp4 GTAGCAATGAGAGACTGACT 2323
Foxp4 GTTCCTTCCTTGCTCCCACA 2324
Foxp4 GAGTTAATAAAGCCTCCCAT 2325
Foxp4 GCCTCAATTAGGACAAGATG 2326
Foxp4 GACTGAGTAGGCCTGAGTAG 2327
Foxp4 GTGGGCCAGGAGCTGAAAGG 2328
Foxq1 GAGAATTCATTCACCTTCTA 2329
Foxq1 GGCCATAGAGAGGAAGTAAG 2330
Foxq1 GGCCGAGGGACTGGTTGCAT 2331
Foxq1 GACAAAGCATTGATTTGGCC 2332
Foxq1 GATGGATTGATAAGTGCCTG 2333
Foxq1 GCCTAACCAAGATCAAGGTA 2334
Foxq1 GCACGGGTGTCAAACAGGAA 2335
Foxq1 GAAGCCGGCTAAGAAACAAG 2336
Foxq1 GAAGCTGGCGTGGTAGGCAT 2337
Foxs1 GCTGCCCTGAGCCTGAGCTT 2338
Foxs1 GTTCTGTCCTCAGGGCAGAC 2339
Foxs1 GTACTGGGAGTTCTGTGAAC 2340
Foxs1 GTACCAGCACCAATACCTAG 2341
Foxs1 GCCTGAATAGTATAGCCCGG 2342
Foxs1 GAAGGAAAGAGAGAGAGAGA 2343
Foxs1 GAGAGTGGGAGACACAGCAG 2344
Foxs1 GTAGACTTTGGAGGGCTACA 2345
Foxs1 GATAGTTGTGTGGAGATGGG 2346
Gab1 GAGTTGACTGATGTGATGCT 2347
Gab1 GGTAGAACAGCTCCTGGGTC 2348
Gab1 GGAGAATTCACCCTTCAAGA 2349
Gab1 GTTCCTCTCTGGCTGCCTCG 2350
Gab1 GTGTGTTTGAAACAAAGCCT 2351
Gab1 GCTTGAGTGAGTTCTCCTCC 2352
Gab1 GACCTCTTCCTTAAAGCATA 2353
Gab2 GCTGGCTATTAATTCCTCTT 2354
Gab2 GAGTTAACTTACAGTGAAGC 2355
Gab2 GTAGATCAAAGGGTGCTTGG 2356
Gab2 GCTTCGCTAGATTGTAATTT 2357
Gab2 GAGGATTTGTGGCCAGCAGC 2358
Gab2 GCGCTCTCCCATAGTGCCTC 2359
Gab2 GCCATCTCCTCCACAAAGCC 2360
Gab2 GGACTCATTTCAGCCAGAAT 2361
Gab2 GCATGTCCTTGCAGGGCTTA 2362
Gab2 GAGTGTGGCTTTGAATGTTA 2363
Gabpa GATGGCGGAGTCTTAGCTGA 2364
Gabpa GCCTCCTGGGACTGAGCTTC 2365
Gabpa GGGTTGTGTGGCCTTTATCA 2366
Gabpa GGATATCATAGAAGTGCGGT 2367
Gabpa GCTGTGCTACAGTGCTTACG 2368
Gabpa GAGTACGCTAAATTAGACGT 2369
Gabpa GCGATCTGTTCATGATCACA 2370
Gabpa GACAGAAGCCAAACAGGAGG 2371
Gabpa GGACTCAGTGCAAGGTGACT 2372
Gabpa GGTTTCTTCACGAGGAGAGA 2373
Gabpb1 GAGACAACCGAGAATAACCT 2374
Gabpb1 GGCCAGTAGGCTGATGTCTA 2375
Gabpb1 GCAAAGGGAGAGGACTGCTA 2376
Gabpb1 GTTCATTCCTTCAGCAGTGC 2377
Gabpb1 GGTGAAGGCCATCCAGTCGA 2378
Gabpb1 GTACCCGGAATCGCTGGTTG 2379
Gabpb1 GGCATGGAGAAGCAGTAGTT 2380
Gabpb1 GTTAGGTTTGGTTGGTTGGT 2381
Gabpbl GATTTAACTTACCGTGGTCC 2382
Gata1 GTGTATCTGAAGTTTGTTAC 2383
Gata1 GAGGGCTAAATATAATCCCA 2384
Gata1 GTCAGTCGGACCACTTAACA 2385
Gata1 GTGATCTTATCCCAATCCTC 2386
Gata1 GCCCTGGAAATCCGTAGGCT 2387
Gata1 GCAAGGGTGAGAATTGGAGG 2388
Gata1 GTGCCATTGGTGTGAGGATG 2389
Gata1 GCCTAGCCTACGGATTTCCA 2390
Gata1 GTGGAGGGACAATGGCTGGT 2391
Gata1 GATCCTGATACTAATAGCAG 2392
Gata2 GCAGTATGAGGCCCAGAACT 2393
Gata2 GCGTCTGATGCGGGTCTGCT 2394
Gata2 GTTCTTTGAATTTCTCAGAG 2395
Gata2 GAGGTTCCTAAGATACCTTC 2396
Gata2 GAATAACCGTTCTAATGAGG 2397
Gata2 GACCACCAGGCTGAACTGCC 2398
Gata2 GCTGAATAACCGTTCTAATG 2399
Gata2 GTCGTCCGTAGCAGTGGAGG 2400
Gata2 GGAGTCAGTTGGATTTGGGC 2401
Gata2 GTCCGTAATTGGGTAACTGG 2402
Gata3 GGTGCAGGGIGAACTCAGAA 2403
Gata3 GGACGCCCGCGTTATTGTTA 2404
Gata3 GGGCTTCCTCTTCCCTTGGC 2405
Gata3 GTAGCAAAGCCGATTCATTC 2406
Gata3 GGCACTGGATCCAGCCTGTA 2407
Gata3 GGTCTGAGGTAGTTTAGGGT 2408
Gata3 GCTTGGCTTTAGAGGGTTAC 2409
Gata3 GTCTCTGGGACAGGGTCTGG 2410
Gata3 GGGACACGATCCTCAGCACA 2411
Gata3 GTTGCAGTTTCCTTGTGCTG 2412
Gata4 GATTCTGACTGGCATTGTTT 2413
Gata4 GCAGTCAGTCCTCGAACCTA 2414
Gata4 GTCTCTAGGCACTGACCTTA 2415
Gata4 GTTTCCAATACAAGATTAGA 2416
Gata4 GAAAGCTACAGACTTAAGGC 2417
Gata4 GGGAGGCCAAAGAGAGGAGG 2418
Gata4 GAAGGAAAGCACTCAGTGCC 2419
Gata4 GCACAGAGGTCGCCTAGTTC 2420
Gata4 GCAACGCTGAGGATCAGACT 2421
Gata4 GTGCCATCCCGAGCCTTCTC 2422
Gata5 GATGGAGCTAGAAGGACCTA 2423
Gata5 GTCAGTGCCCAGGTCTAGAC 2424
Gata5 GGGAGCCTCAGCCAGTCTTT 2425
Gata5 GTCCTCCAACTTGGCCACTC 2426
Gata5 GCATTGACCAGTGGGCAGCA 2427
Gata5 GGATTAAACTCAGTTCAGAT 2428
Gata5 GGGTGCTGCAGACAGATACG 2429
Gata5 GGACCGACTAGAAGAGAGAA 2430
Gata5 GGGAGCTCGGAATAGACGTG 2431
Gata5 GGGACCGACTAGAAGAGAGA 2432
Gata6 GAAGGTGGCACACCAACCTA 2433
Gata6 GATAACGCGTTGAGAAGGAG 2434
Gata6 GTATATCACTGCTGCTGCCT 2435
Gata6 GCTAAAGGACACCAAGGGAG 2436
Gata6 GAACGGTTTATAGACCTACT 2437
Gata6 GTTACAGCGCTGGATGATTA 2438
Gata6 GGCGAGGTAGGGAATACACA 2439
Gata6 GAGAAGGGAAATGACTTACT 2440
Gata6 GTCAGTTACTAGCAACTGCG 2441
Gata6 GACCTGAGCATCCCGAAACA 2442
Gbx1 GCTTCCTCTCTCAAACACAG 2443
Gbx1 GATTGATGAGGCCGGACCCG 2444
Gbx1 GGACTCGGTTCTCTAAGCTC 2445
Gbx1 GTAGCTAGTATCATGTGTTG 2446
Gbx1 GAATACCTGCCCAATCAGAA 2447
Gbx1 GACCTCACTGTAAATGGAGG 2448
Gbx1 GAGGACAGAGCTGGGCTGAA 2449
Gbx1 GGACAGTCAAGGCGGAATGG 2450
Gbx1 GCTTTGTGAAAGTTTGCGGC 2451
Gbx1 GAGCCAGGGAGATAGAGTGA 2452
Gbx2 GATTGCGCCGAAAGGAAAGT 2453
Gbx2 GCCTCTCATGAGGCCTCCAC 2454
Gbx2 GAAGCTTCGGTCTGAGCAAG 2455
Gbx2 GGCTGGCGGTGAAAGGGAAG 2456
Gbx2 GAGCAGGGATCGCTCACGGA 2457
Gbx2 GTATTATTATTACCTGGAGG 2458
Gbx2 GAAAGGCACTGGCCAGCAGG 2459
Gbx2 GTTGCGGCACACACTGTCCC 2460
Gbx2 GTTATTTCCCAACTATGGCC 2461
Gbx2 GCGGCTGAACTTCCCTGGTG 2462
Gcm1 GTTATGAATGCCACAGAGAG 2463
Gcm1 GAGCTTCAGACTCTTGGACT 2464
Gcm1 GGTAAGGTCAGCTACTCCAC 2465
Gcm1 GATGAGGCATGGCAGAACTG 2466
Gcm1 GGAATCCCAGGTAGTCTGCT 2467
Gcm1 GCTTTCAGCCAGGGACAGGT 2468
Gcm1 GGAGTGTCTCTGCAAATTCA 2469
Gcm1 GTCACCCATAGCATGCCTGA 2470
Gcm1 GTCTAAAGGTGAGACTGGAA 2471
Gcm1 GAGATGGCTTTCAGTGTTTC 2472
Gcm2 GTCACTCTTAGACTCAGCGG 2473
Gcm2 GCAGGTCACTGTTAGAGGAG 2474
Gcm2 GCAAATCTGAAGGTTGGGAC 2475
Gcm2 GGTTGAGGCTCTGGAAGCAA 2476
Gcm2 GCTAAGGCTGACAATGGAAT 2477
Gcm2 GCTGGTGCGCTTTACAGCCA 2478
Gcm2 GTTTCCAACTTGGTCTGTTT 2479
Gcm2 GAAGTTAAGCAGTAGGCAGT 2480
Gcm2 GGTTTCCAACTTGGTCTGTT 2481
Gcm2 GTGACCCAAACAGACCAAGT 2482
Gfi1 GAAATGAGATCCTGGAGGAC 2483
Gfi1 GTGAGCAGTTTAAGTGCTGC 2484
Gfi1 GCGACGAACAGAAGCGAAAG 2485
Gfi1 GCAGAAAGAAACCTGCGCCT 2486
Gfi1 GGGAGCATAGGAGGAAGGCA 2487
Gfi1 GCATAGGAGGAAGGCAGGGA 2488
Gfi1 GAAGGCAGGGAAGGGAGAAG 2489
Gfi1 GGACTATGTGCTGCAGTGGC 2490
Gfi1 GACGAACAGAAGCGAAAGAG 2491
Gfi1 GTTTCTCCTGGGACAAGTGT 2492
Gfi1b GAGCATGGAACTTTGGAACA 2493
Gfi1b GGTTTGGGTCAGGGCTGTAT 2494
Gfi1b GGACTGGACCAGGAGTTCTC 2495
Gfi1b GATGGATGCCTCCAGAGATG 2496
Gfi1b GTTTGAGGCCTTTCTGCTGA 2497
Gfi1b GATTGAATACCCAAGTACCA 2498
Gfi1b GTGTAGCACAACCAGTTCAA 2499
Gfi1b GAAATGCCCTGCGCTGGCCT 2500
Gfi1b GAACATGGACCCAGATGTGG 2501
Gfi1b GCTTTAGACAAGGAACCGGT 2502
Gli1 GTCACAGTAGAAACAGATAA 2503
Gli1 GTGCGACCTATGGAACATGA 2504
Gli1 GTATAGGGTCCCTCAAGGGA 2505
Gli1 GTGGTCCAGGGCTGGAAACT 2506
Gli1 GGCAGTATAGGGTCCCTCAA 2507
Gli1 GGTGACTGGACACAGAGAGA 2508
Gli1 GGATATACGAGGGAAGTGAG 2509
Gli1 GATATACGAGGGAAGTGAGC 2510
Gli1 GGGTGGATAGAAGCTAGAGA 2511
Gli2 GGTTTGTTTACATGTATTGG 2512
Gli2 GGTAACAAGAAAGGAAGAAT 2513
Gli2 GATCCAATCAGTGAGTAACA 2514
Gli2 GGGTTTGTTTACATGTATTG 2515
Gli2 GAAGGAGCTTTCTCTAAGGC 2516
Gli2 GTCCACTCCAAGAAGCAAGC 2517
Gli2 GAACAACCAGCGGAGGGCTG 2518
Gli2 GCTACGGCGCACAGAGGATC 2519
Gli2 GTAGCTGGAACTTTCTGGTA 2520
Gli3 GGCCTGGATGTGTCTGTGTG 2521
Gli3 GAAACATCCTTCACTCATCT 2522
Gli3 GATACATTGITTCTGGCATT 2523
Gli3 GTTATCTCTTAACTCAGTAG 2524
Gli3 GGAAGTTTCAGGCTTGGCCT 2525
Gli3 GAGAGGTGGGCAACTCAGAT 2526
Gli3 GTTCTGATTTGGTTCAACCG 2527
Gli3 GTTCTGATAGCGTGGTGGGA 2528
Gli3 GAAACAAGAGGAATTCTTGA 2529
Gli3 GTGAACATGGTTTACAGAAA 2530
Glis1 GGAATGGGTACAGGAGAACG 2531
Glis1 GCCTGAACTCTCCATTCAAT 2532
Glis1 GGCCTGGGTTCAGATGACAA 2533
Glis1 GGCAGCAGGGTCTCAACTGT 2534
Glis1 GAAGACACTGGCGTGGGAGT 2535
Glis1 GTCTGCTACAGCAGGTAGCC 2536
Glis1 GTGTGTTTCTGCAACCGGCC 2537
Glis1 GTGCAGGCGATGAGCTGTTA 2538
Glis1 GGGTCCACACTTTAGAATTG 2539
Glis1 GTAAATGAGTTTGTTGCTGT 2540
Glis2 GTCCTGGATCTGGACTGGGC 2541
Glis2 GATAATGTCGCAGTGCTGCC 2542
Glis2 GGATAATGTCGCAGTGCTGC 2543
Glis2 GCACATCGGTAGTGTGAAAC 2544
Glis2 GTAGTTCAGCACGTGTTCCT 2545
Glis2 GTGAGATACTGCACTAGGGC 2546
Glis2 GGCCTTGTGCTTATTTCACC 2547
Glis2 GCCTACCTTCGCACCAGACC 2548
Glis2 GGTCCTGGATCTGGACTGGG 2549
Glis3 GCTAAAGTTCCAAGCATCAC 2550
Glis3 GGTAACTGGCATGAAGCTGA 2551
Glis3 GACCACTGGAGTGTACAATG 2552
Glis3 GCTGGAATAAATTCCATGTG 2553
Glis3 GGACCTGTTGTCAACTCTCA 2554
Glis3 GAAGGTGATGGCCAAAGGTA 2555
Glis3 GGTACAGIACGAAGGCCAGG 2556
Glis3 GTCAAGAGGGAGACACTGGC 2557
Glis3 GGCAAGCTCTCTGAGGTAAC 2558
Glis3 GCTAGCTAAAGTGAACAATG 2559
Gm4736 GTGACTGAAGTACTATATAG 2560
Gm4736 GGCCTACAAAGTCATCATGA 2561
Gm4736 GGAAGGCCACAGTCTTTATA 2562
Gm4736 GGCCACAGTCTTTATAAGGA 2563
Gmeb1 GTCAGCTCGAAGGAGCACAT 2564
Gmeb1 GGCAGTGAATGGAGCTTGTA 2565
Gmeb1 GTGGAGTCCTCTCTGAGGCT 2566
Gmeb1 GGTCAGCCACTGGGTTCAGC 2567
Gmeb1 GAAAGCCTAGGTCAGCCACT 2568
Gmeb1 GTGTGAGGAGGGAAGTAGGT 2569
Gmeb1 GATGTCCTCTGTAAAGGATG 2570
Gmeb1 GGCAATGTGGTCAGGCCTTG 2571
Gmeb1 GTGGTGGAACTCAGGTGGTC 2572
Gmeb1 GGTGGGTAAGTGCTCTGACA 2573
Gsc GAACCTATCGGCACCCACGC 2574
Gsc GTGAACAGCCTCTTCCTTCT 2575
Gsc GTTTGCCAGGTGGCAATGTT 2576
GsC GTTAGGAGCTAGGGAGAGTC 2577
Gsc GCGCAGAACTAGGCAGTGCG 2578
GSc GCCACTCAATATGTTGAGAA 2579
Gsc GGGTCCGGGAGCTTCTTTCT 2580
Gsc GATAGAGACCGGCTTCAGTT 2581
Gsc GGAGAGATGCCAAGAGGAGG 2582
Gsc2 GCCAAGTATTTGTTCTCAGT 2583
Gsc2 GCAGCCATTCTGTAACCATG 2584
Gsc2 GAGGGAATGAGGGAAGCCAG 2585
Gsc2 GGAGGGAATGAGGGAAGCCA 2586
Gsc2 GCCAGGCTCTGTGCACTTGG 2587
Gsc2 GAAGCCCATAGAGTCCTCAC 2588
Gsc2 GCACCATGTCATCTTCCTAC 2589
Gsc2 GGACTTGGTAAAGTGGGAGA 2590
Gsc2 GGGATTAGCACGCGCGAACG 2591
Gsc2 GGGATTAGCACGCGCGAACG 2592
Gsx1 GAGCAATTAGAACGGGAATT 2593
Gsx1 GGGAGTGAGAGCCGAATTCG 2594
Gsx1 GTTGCCAGCGCCTTCTCTTC 2595
Gsx1 GGAACGCAGAGGCAGAAGGC 2596
Gsx1 GAAGCTGTGTACACAGAGCG 2597
Gsx1 GAGAGAAGAGACTCCACAGG 2598
Gsx1 GATCGCCAGCGCAAAGCCAA 2599
Gsx1 GTAACAGAAAGAAAGGGACC 2600
Gsx1 GGAAGAAGTAACAGAAAGAA 2601
Gsx1 GAGTGCACCGGCGTGTCTAG 2602
Gsx2 GCCGAATAAATCCTTCCACG 2603
GsX2 GAGGGAGAAGACAGATATAG 2604
Gsx2 GAGCTCTAATTGCCAGGACT 2605
Gsx2 GTGGTCACAGAGATGGAAAG 2606
Gsx2 GGGCAGGGAACAGCAGTTGG 2607
Gsx2 GAGAGTGATGGAGGGAGAGG 2608
Gsx2 GCCTACCTTCCTCCCTCGCT 2609
Gsx2 GAGAGTAGGTTGGTCGGAGC 2610
Gsx2 GCTGGTTAGAAAGATGCACA 2611
Gsx2 GGTAGGTTATCTACAGTCCT 2612
Gft2a2 GCGTGAAAGGCTTCAGTGTG 2613
Gtf2a2 GGTTGGTATCAGTCTCCACC 2614
Gtf2a2 GACTGCAGTGTAGGGAAACC 2615
Gtf2a2 GCAGCTATAGGTACTGCAGA 2616
Gtf2a2 GCAAGAGGTGCCAGGAAGTG 2617
Gtf2a2 GTTTACCAGCCGTGAAGGGT 2618
Gtf2a2 GAGCCAAAGTATAACAGAGA 2619
Gtf2a2 GTCTATATACAAAGGTACCA 2620
Gtf2a2 GGTAGCTGTCAGTTACTCCA 2621
Gtf2a2 GAAACAGATCACGTATGGTG 2622
Gtf2f2 GTCCTGACGTAGTCGTGCGC 2623
Gtf2f2 GTTTGAAAGAGGCTCTGAAA 2624
Gtf2f2 GTGTAAAGATCAGGGAAAGC 2625
Gtf2f2 GCAGGTGGATGGGCTTGGTG 2626
Gtf2f2 GCATCACACACTATCATATG 2627
Gtf2f2 GCAGTAAGGTATTGGAAGAA 2628
Gtf2f2 GAACCGTGCGTTTACAGCAA 2629
Gtf2h1 GGTGGAAGCAAGAAGGCACG 2630
Gtf2h1 GCCCAGTATGTAAAGATCTT 2631
Gtf2h1 GATGACAGGTGGAAGCAAGA 2632
Gtf2h1 GTTCAGGATAGCTGAATAAT 2633
Gtf2h1 GTTCTTCCGCTGGGAGGGAC 2634
Gtf2h1 GCCTTCGGGCAGTAGATTAA 2635
Gtf2h1 GCCAGCGTTTGTTAGGAGGG 2636
Gtf2h1 GCCTCACTTCCTTCGTTCTC 2637
Gtf2h1 GGTAAGTTGAGACCGAAGAA 2638
Gtf2h1 GGCGTGATCGTCACGTGACG 2639
Gtf2h2 GCCAGGCTTGCTCTTTGCTT 2640
Gtf2h2 GTTCTCTTGAACACAAGGAA 2641
Gtf2h2 GACAGATCACCTCCCACATG 2642
Gtf2h2 GAGGGCAACTACGTATGGTG 2643
Gtf2h2 GACTGCCGGTACTTCCGGTG 2644
Gtf2h2 GTTCACCAATATTTCTGCTG 2645
Gtf2h2 GTGTAGACAAGTGTGAGACC 2646
Gtf2h2 GGGAGGTGATCTGTCCTGCC 2647
Gtf2h2 GCTGCCAGAAGAGGGAGCTA 2648
Gtf2i GGCCTGCTGGAGAAGGAAGG 2649
Gtf2i GTTCATGCCGCAAGGCTGTC 2650
Gtf2i GGGTTCAGAACTACAACTCC 2651
Gtf2i GTGGCCTGCTGGAGAAGGAA 2652
Gtf2i GTTTACTTTCTTTGTAGCTG 2653
Gtf2i GTAAACTTAAGACCCTCCTC 2654
Gtf2i GAGGGCGCCCGAATATTCGG 2655
Gtf2i GGCGGACATAAGCGGTGGGA 2656
Gtf2i GGACAGGCAACGGATGGGAG 2657
Gtf2i GTCGCCTGATTTGCAGAGGG 2658
Gtf2ird1 GGGATCAGAAACAAGGCCAT 2659
Gtf2ird1 GTAGCTGGCAGAGAGGCTAT 2660
Gtf2ird1 GGACAGGATCAGTAGAGGGA 2661
Gtf2ird1 GGCTAGGCCTTTGCTGGGAT 2662
Gtf2ird1 GTGTCCAAGGTCAGAAGGGA 2663
Gtf2ird1 GATGAGGGATGATGGAGATG 2664
Gtf2ird1 GTAGAGGGAGGGAGGGAAGG 2665
Gtf2ird1 GGGACAGGATCAGTAGAGGG 2666
Gtf2ird1 GTAGTATACAGGAGGTCAGA 2667
Gtf2ird1 GATCTAGAAGGAGACCAGGT 2668
Gzf1 GGTAAAGCAATGATTTACCG 2669
Gzf1 GGTGGGTCAAGTCTTGGCGT 2670
Gzf1 GCAGAGCTATTTGACAAAGT 2671
Gzf1 GTGTGAGGGACAAAGCGCTG 2672
Gzf1 GTGTTATGGAGCCAACCACA 2673
Gzf1 GCCTGAGTCTCCCAGTGTGA 2674
Gzf1 GGAGACTCAGGCAGCCACTG 2675
Gzf1 GCTAAGGCGCAACCAAAGGA 2676
Gzf1 GTGGGTCAAGTCTTGGCGTA 2677
Hand1 GAGGTGGAAGTGGGAGGGAA 2678
Hand1 GTAACTTAGGAGACTGAAGC 2679
Hand1 GTTGTGCAAGAGATTGTGAG 2680
Hand1 GTGTAAGACAATTACCAGGC 2681
Hand1 GTTCAGTACAGGGAGTGAGC 2682
Hand1 GAAGTGGGAGGGAAAGGGAG 2683
Hand1 GTGAGTGTCCATTGTCCTTG 2684
Hand1 GTGATCTGGGATCTCAGGCA 2685
Hand1 GGGCACTGACCAGTTTGTTC 2686
Handl GTGGGAGCCTGAAGGCCATT 2687
Hand2 GCCAGGTAAACTTGCTGCTT 2688
Hand2 GCTTGTACAGCCCAAGAGTG 2689
Hand2 GGCTGTACAAGCAGGCCCTC 2690
Hand2 GTCTGGAAGGCCACATCAGA 2691
Hand2 GTAGCTGGACCTAGTCTTGC 2692
Hand2 GGACCTGAGGAGGCAAGCAG 2693
Hand2 GTACCCTGGGAGCAAGAAGA 2694
Hand2 GAAGAAGGTCCCTGTGTAAT 2695
Hand2 GTGCTGTCAGTGAGGAGTGA 2696
Hand2 GTGATTATGAGGGAACTAAC 2697
Hbp1 GTTGCATCATCAAAGATTTG 2698
Hbp1 GTATCTGAAAGTTGTACACT 2699
Hbp1 GGTGCTGAAATACCCAACCA 2700
Hbp1 GTTTCTCTTTCTACTTTGTT 2701
Hbp1 GGCCTAGAGCGTCCTTGGTT 2702
Hbp1 GTTGGCGGCGTATTGAGTCA 2703
Hbp1 GCCAAGTGCCATGTACTGTA 2704
Hbp1 GGCTGTGTCTCAACTAATTC 2705
Hdac2 GTTGGACACAGTTTCACAAG 2706
Hdac2 GGAAGAAGACTAGCATGAGT 2707
Hdac2 GGGAAGAAGACTAGCATGAG 2708
Hdac2 GAGTAATTCTAAGTCTCTTG 2709
Hdac2 GGTTGGGTCAGGGACCACAG 2710
Hdac2 GTGTTTATTACGAGCAGGTA 2711
Hdac2 GATAAAGTAGACAAAGCACG 2712
Hdac2 GGAGTAATTCTAAGTCtCTT 2713
Hdac2 GGTAGCGGGTGTGTGTGTGG 2714
Hdx GCACTTATCTGCTAAATCTG 2715
Hdx GCAATCACCTGTGAATTACA 2716
Hdx GGAAGAGGCAGCCCTACTAC 2717
Hdx GGACCCAGTTTGAGCACACT 2718
Hdx GTTGTACACTTACTTTGTTC 2719
Hdx GAATATGGCAAAGTGAAAGA 2720
Hdx GTAGTAGGGCTGCCTCTTCC 2721
Hdx GAAAGCAAAGTACAAATTGT 2722
Helt GGGAGAGCTTCTGGAGACGG 2723
Helt GCTGTGAGATGCAGGACTTC 2724
Helt GGATGTCCGGACAAATAAAG 2725
Helt GTTAGACAGTGAGACTGGGT 2726
Helt GCAGCACCTAGGAAGCTCCG 2727
Helt GTAAATCACCCGGAGATCCA 2728
Helt GTATATTCACTCGCACACAA 2729
Helt GTGCCTGGAGGGTGTGGAAT 2730
Helt GGTATATTCACTCGCACACA 2731
Helt GAAGTTGATCCTCTTACTGT 2732
Hes1 GGCTTTCTGGACAATGCTTG 2733
Hes1 GTTCTATAACTGAGGACATC 2734
Hes1 GAGAGGAAGGGAGCTACCGA 2735
Hes1 GCAGTTTGACATCAGCCGGC 2736
Hes1 GCTGATGTCAAACTGCAGCT 2737
Hes1 GATATATATAGAGGCCGCCA 2738
Hes1 GAGAGGAATGAATGGGCTAG 2739
Hes1 GTAAGGGCATGTTTAGCGTG 2740
Hes1 GGCTCCTAAGTGGCACAGGT 2741
Hes1 GCTTCTAGTAGGGCTACTGG 2742
Hes2 GGTTGTTCGGGTCTCGCCTT 2743
Hes2 GTGCTTGAGGAGCGGAGCCA 2744
Hes2 GTCTTTGATCAGTGTAGGGT 2745
Hes2 GCTTGTACAAAGTAACTCCT 2746
Hes2 GCTCCATTGAGGGCTTTGGT 2747
Hes2 GTCACATGACAGACGAGTGG 2748
Hes2 GGGACACTGGACTGAGTTGG 2749
Hes2 GATCAGTGTAGGGTGGGCTT 2750
Hes2 GCGTCTGTCAGGAGCCTTTC 2751
Hes3 GACAGACTCATCACTGCCCT 2752
Hes3 GCACAAACTGGTATGGGTGC 2753
Hes3 GAAGCCCTGAAATGACTCAG 2754
Hes3 GACTGGGACGAGAGCTTCCT 2755
Hes3 GGCTTCTTCCCTTCCCGCTC 2756
Hes3 GTGTGGTTTGACAGGGAGCA 2757
Hes3 GGGATACAGTCACACAGAGA 2758
Hes3 GAGCTCCGAGGAATTCTAAG 2759
Hes3 GCTCAGTGGTTAGCACATTC 2760
Hes3 GAAACCCTGCTTATGCAAAC 2761
Hesx1 GAGAGATACACGTTTACATG 2762
Hesx1 GCAACAGGGACTGAGCGAGC 2763
Hesx1 GAATATGAGAGTGCAAGTGG 2764
Hesx1 GGCATTTGACAAAGCTTTGC 2765
Hesx1 GCACTCTGTGTTAATAACAC 2766
Hey1 GTGGATGGAGAACTGGACCT 2767
Hey1 GTCATCTGCAGCTCAGAAAG 2768
Hey1 GGGATTGCAGGCTCCAAGAG 2769
Hey1 GTGATATGAGGCTCTGAAGA 2770
Hey1 GCAGATTGGCAGCCGCATGG 2771
Hey1 GTGTTAGATGGAGATGTAAT 2772
Hey1 GATAAGGAGAAGGGAGAGAA 2773
Hey1 GCACCTTCTGATAAGGAGAA 2774
Hey1 GAAACATGGGATGGCGTCAA 2775
Hey1 GGGTGCTCCGTCACTTTAGG 2776
Hey2 GCACACACCGGAGAAACTGG 2777
Hey2 GTGAGCGTGTGTGACGTCTA 2778
Hey2 GACACAGAAACTGGAGGGAG 2779
Hey2 GGCTGTCTGCTCTGTCCCTG 2780
Hey2 GAGTTCAAAGTTCTCGGATT 2781
Hey2 GCGTGTGTGACGTCTAGGGT 2782
Hey2 GGTGTGTTTAGACAGGAGAC 2783
Hey2 GCTGCACACACCGGAGAAAC 2784
Hey2 GACTGGACTGGGCGCAGATT 2785
Hey2 GCAAATCACAGGATCATCGG 2786
Heyl GAAATGCCTAGTGCACACAT 2787
Heyl GGCAGGGAGATGGTGGAGGT 2788
Heyl GTGCTATGCTGTCAGTTCAG 2789
Heyl GGCAGAAGAAGAAGGAGAGC 2790
Heyl GACTGAAGAATTACTTCCAA 2791
Heyl GAGCCTTCGGCTTCTCTTTC 2792
Heyl GGCACCAGGGAGAGGAAGAG 2793
Heyl GTAGGGTGTGGTGGTTGGTG 2794
Heyl GTGCAGAGGGAAGCTGAGGG 2795
Hhex GAGTTGGGCAGTTTCTGCTA 2796
Hhex GAGAAGCGATGGGACTCTGC 2797
Hhex GACTGCGACCGTCGAAGAGG 2798
Hhex GCAGTGTTCTTCGATCCAAT 2799
Hhex GGCTTAGTAGTAAGGGTTAC 2800
Hhex GTGAACTACTGGAAGGTTGC 2801
Hhex GGCCAGAAGGCTGCGCTTCT 2802
Hhex GATTCCGTTAGCATCCAGGG 2803
Hhex GAATCTGAAGCCAGCGCCAT 2804
Hhex GTTCGTTTCCTGCTTCCACC 2805
Hic1 GACCGGCAAGACAGACCGAC 2806
Hic1 GTGTCTTCCCTAGAGGACTC 2807
Hic1 GTGTGGAGCATGCAGGACGG 2808
Hic1 GGGCTCAATAGCTTGGCAGA 2809
Hic1 GTGGTATCCTCGCTCTCTCC 2810
Hic1 GGGACTCCGGAGTGAGGATG 2811
Hic1 GCTCTCTCCTGGTGTGTGTG 2812
Hic1 GAGTGAATAAACACAGAACG 2813
Hic1 GTAAGTGGATTAGATGGAGG 2814
Hic1 GACCACCAACAGTCGGAGAT 2815
Hif1a GCCATAAATAGATACCACCA 2816
Hif1a GCAGTCCTGTCAAGGTCTGT 2817
Hif1a GACACAACTGAGTCTGAATC 2818
Hif1a GTAAGGTCTGCAAAGTGAGT 2819
Hif1a GGCACTTTAACAGTTGAAAC 2820
Hif1a GCTGAGAGCAACGTGGGCTG 2821
Hif1a GCTCTCAGCCAATCAGGAGG 2822
Hif1a GTTGCTCTCAGCCAATCAGG 2823
Hif1a GTTGTGCAGATTGTGAAATG 2824
Hira GCGCATTTATTAGAAGAGCG 2825
Hira GTGTCTGACGTGTGCCTGGC 2826
Hira GGAACTTTGGATGCTTTCTT 2827
Hira GGTCTGGGATTCCGAGAGGC 2828
Hira GAAATCTGCTTGCTAACCCA 2829
Hira GAAGTGAACGTGCTGAACTA 2830
Hira GGGTGATGCTGTGTGCTGCG 2831
Hira GTCTGCCGCTAGATGCATGC 2832
Hira GTCCACTGTCTTCCCGAGGA 2833
Hira GGGCGCATTTATTAGAAGAG 2834
Hivep1 GGGCGTGAGAGGAAACGCTG 2835
Hivep1 GGGCTGGGTTGTTGACTTGG 2836
Hivep1 GTGGGCGTGAGAGGAAACGC 2837
Hivep1 GCTTAGGCTCTGGGAAGCAC 2838
Hivep1 GGTTCAAACAGCTCGGCTGG 2839
Hivep1 GCTTGGCTTGGGAAGAGCCC 2840
Hivep1 GAACTTTGGAAGCCGAAGAG 2841
Hivep1 GGAACTTTGGAAGCCGAAGA 2842
Hivep1 GGAATAACCTTGGCTTTCCT 2843
Hivep1 GAGAGCATCGGTCCAACCCG 2844
Hivep2 GAAGTTCTCTGATCCTACAA 2845
Hivep2 GACTCGCCAGTGTTTCTGCG 2846
Hivep2 GAACGCTCGAATCCAAAGAG 2847
Hivep2 GAAGGGAATCCCAAGCGAGT 2848
Hivep2 GCGAGAAATCCTTGGTACGC 2849
Hivep2 GGCTAGAGAGGGAAGGGAAT 2850
Hivep2 GAGAACCAGAAGCGCGCAGC 2851
Hivep2 GGACTCGTGTGCACCCTCAA 2852
Hivep3 GTGGGCTTCAGAGTGCATGA 2853
Hivep3 GGAGAAACATATGCAAATAC 2854
Hivep3 GTTGGATCAGAATGAGGTCA 2855
Hivep3 GCGGTCTTGACGTTGAGCGC 2856
Hivep3 GAACCTCCAACTTAACCTCT 2857
Hivep3 GGGATTAAGCTGGAGGTGGA 2858
Hivep3 GTAGTTGGCATGCACAGTTT 2859
Hivep3 GTGATGGAGGAGCCTGCTGA 2860
Hivep3 GTATTGGAGAATAGCAGCCT 2861
Hivep3 GGATCCCTAGCTATTGAAAG 2862
Hltf GTCAGACGCTCCCTATCTGA 2863
Hltf GGCTTCTTGAGTGAGCCACA 2864
Hltf GCTCAAGGTTCTGACGGACT 2865
Hltf GTATGCGAGACCCTGAGTTC 2866
Hltf GCTAAGAATAAATAGAGTCA 2867
Hltf GTTCACGAGGTGAAGGGCTG 2868
Hltf GAGGCACCAATGCATTGTCG 2869
Hltf GAAATGCAGGTATCCCACCC 2870
Hltf GTAAGGTCCGAGGTGGTGGC 2871
Hltf GTGGTGTGGACACGTCTCAC 2872
Hlx GATGTCCCAGTATCAGGGAC 2873
Hlx GCTATGATGTCCCAGTATCA 2874
Hlx GGCTACTATCAGCTCAGGAT 2875
Hlx GTAGACTTGGGTCGGGATTC 2876
Hlx GGCTATGATGTCCCAGTATC 2877
Hlx GTCTAGCAGGGAGCAGAGGG 2878
Hlx GCCTGTGGTCTGTTTGGGAG 2879
Hlx GGGAGCTCCGATTAGGCCTC 2880
Hlx GCCAAAGCGACTGGTCTACA 2881
Hlx GTTGCGTTGTGCACCTAGTC 2882
Hmbox1 GTCTAGCATCCATGGTATTC 2883
Hmbox1 GCTGGAAGCTGTAGTTCCCT 2884
Hmbox1 GATGGAAAGGAAGGATGAAT 2885
Hmbox1 GCGGCGGCGATGAATTTGAG 2886
Hmbox1 GACTTTCACAGGTGCACATG 2887
Hmbox1 GTTTCCACTACTAAGTCAGA 2888
Hmbox1 GTTTATTCAAACCCTTTGGT 2889
Hmbox1 GAAGACCTCCTGACAGATGC 2890
Hmbox1 GAATCTTCCTAATTGCTACG 2891
Hmga1 GTGTTTGCCTACTTCTAGAG 2892
Hmga1 GGATACCCTTCCTTCCTGGA 2893
Hmga1 GGCGGCCCTGCTGTTTAAGT 2894
Hmga1 GGTTCGAGTTTCCCGCCTCT 2895
Hmga1 GAGATCCCAACTGGAATGTC 2896
Hmga1 GGGCACAAAGATGGAGGGCG 2897
Hmga1 GCTCCTTTGAAGCCTGCACC 2898
Hmga1 GTTGCAAGGAAGTCCTGTTC 2899
Hmga1 GTAGGAGATGCAGGAAGCAC 2900
Hmga1 GAAGACCAGACAAGAGGCAG 2901
Hmga2 GAAGTTTCCGGAAGCATTCA 2902
Hmga2 GAGTTCTGAGTCTTCTCATT 2903
Hmga2 GTTATGGGCGTCCCAGCACG 2904
Hmga2 GGCATTTCTCAGTGGAGCGG 2905
Hmga2 GTGCACGCTTGTTTGTGCGC 2906
Hmga2 GACAGCAGGTGAAGGAGAAA 2907
Hmga2 GCTTGGAGAGGGAAGAGACT 2908
Hmga2 GCGGCACTGCACAGATGCAG 2909
Hmga2 GCACCCAAATTTATAAAGCA 2910
Hmga2 GGTAGAAGCCAAGCTCTCCA 2911
Hmx1 GAAGTCTGGGTTACCCTCTG 2912
Hmx1 GATGGAATGCTCTCATATCC 2913
Hmx1 GGATAGGTGAGACAGAAACA 2914
Hmx1 GCTTGGGAGCACTAGAAAGA 2915
Hmx1 GTCTTACCCAGCACTCCCTC 2916
Hmx1 GGACCAGGCAGACTCTGCTA 2917
Hmx1 GGAGAGCCTTGCTCACCCTC 2918
Hmx1 GATCCAATCGCGCAGATTTA 2919
Hmx1 GATCTGTCAGGAAACCTGCC 2920
Hmx1 GTTGCCTTCTCCTGGACAGT 2921
Hmx2 GATCAGGTAACAGGTGCTCT 2922
Hmx2 GAGAGCACTGACTGGTGTTG 2923
Hmx2 GCGACACTAAGAAGTTTGCC 2924
Hmx2 GGGAGTGAAGTTTGGTCACG 2925
Hmx2 GTGTGGGAAGGCGAGCTGTG 2926
Hmx2 GCATCCTGAAACAGAAAGCC 2927
Hmx2 GGAGTCTGAAAGAGGAGGTG 2928
Hmx2 GTCACCGCATTAACCTCTTC 2929
Hmx2 GGAGCTTTGCTGCTCTGGGC 2930
Hmx2 GGGAGAGGCCACAAGAAGGA 2931
Hmx3 GCCGAGATICICCAGGGACT 2932
Hmx3 GAAAGATAAAGAACGGGCTG 2933
Hmx3 GATTTCGTATAAGGCTTTAC 2934
Hmx3 GCTACTTACAAGGCAATAGT 2935
Hmx3 GCGGGCCTCTGAGGAATAGC 2936
Hmx3 GCCGGAAATCAGACCATAAA 2937
Hmx3 GGAGAGAACTCTTCCAAAGG 2938
Hmx3 GGCCAAGGAACTATCACCAG 2939
Hmx3 GCTGCCTCTTAACTCTTCTT 2940
Hmx3 GACACCTGCAGCATGTCCCA 2941
Hnf1a GCTGGGACAGCAGGAAGCTC 2942
Hnf1a GCTAGAGACCTGCATAGGAA 2943
Hnf1a GGGAGTCATGGCCTGCAATT 2944
Hnf1a GTGGTTGGTGGCACGATTGT 2945
Hnf1a GCCTGTTTCTTTGGGCCGCT 2946
Hnf1a GAGTGAGCAGAAGGGAGGGT 2947
Hnf1a GCAATTGGGAGTGAGCAGAA 2948
Hnf1a GCCCAACATCAGACTTCCCA 2949
Hnf1a GGCAGTTTCCAGAATCTTCA 2950
Hnf1b GATCACCTGTGGGAGGACTC 2951
Hnf1b GCAGTAACTCCTCCAAGGCC 2952
Hnf1b GAAGACCACCTGTGCAAAGC 2953
Hnf1b GGAGCCGACTTAGGGAAGCC 2954
Hnf1b GTGCCTCCTTGCTTCCTCTC 2955
Hnf1b GGACGGCAGTAACTCCTCCA 2956
Hnf1b GAACCTAAGGGACAGTCCAA 2957
Hnf1b GTCTGAAAGCTAAAGGGTGG 2958
Hnf1b GCTCTGGCAAGTCCCAATCC 2959
Hnf1b GTTTGGCTGATAAACAGAAT 2960
Hnf4a GGGTGCCTGCCTTGGAAGAT 2961
Hnf4a GAAAGACCCAAGTGTGGGCT 2962
Hnf4a GAGAACCACAAATCCACTTG 2963
Hnf4a GCAGGACCTTAGGAAGCTTC 2964
Hnf4a GTGAGTTTAGAAACTCTCTG 2965
Hnf4a GACTATTAATGAGCGGGAGG 2966
Hnf4a GTTGGTTTCTGACTGACACC 2967
Hnf4a GTCCTCTGGGAGACTCAGCC 2968
Hnf4a GACTCCCACTAGCTGGAGAA 2969
Hnf4g GACATATTGTTGGACTTGAA 2970
Hnf4g GGCTGTAAACAGCACACCTG 2971
Hnf4g GGGTAAGAACATTAAGGGAG 2972
Hnf4g GACATGCCAATGTTGCAGAG 2973
Hnf4g GATTTCCATCATATGATCAT 2974
Hnf4g GCCTAAGAGATCCAGATGAA 2975
Hnf4g GCATCTGCAGTCCTGCTCCC 2976
Hnf4g GATCCTCTGAGAGCTTTCTG 2977
Hnf4g GTGTTGCAGTCACTGAGGGA 2978
HnF4g GCTTTGTTCTGCAAGAGTTC 2979
Homez GGGAACCAAACACCTGACTC 2980
Homez GGGAAGAGTCTGTGCTTGAA 2981
Homez GAGATCTGAAGGTGACCTCT 2982
Homez GCCAATC3CGGACCTCTGCT 2983
Homez GGAAGGAGATCCACACAATT 2984
Homez GAGTTCGTGAAATGAGGAAA 2985
Homez GTCTTCCGAGGGCCTTCCTG 2986
Homez GCTCTTCTGATTAATGGACT 2987
Homez GGGCTGGGAACATGTCTTCC 2988
Homez GGAAGGACCACAGGATGCAG 2989
Hoxa1 GAGGCCTCCTGGCTCTCTTG 2990
Hoxa1 GTAATTTACGTGTGAGTTTG 2991
Hoxa1 GTCCCTCTACATTCCGAGGC 2992
Hoxa1 GGTGAAGAAAGAGGGCTTGG 2993
Hoxa1 GCCTCCTGGCTCTCTTGTGG 2994
Hoxa1 GAGCATGCTCACTCTAAAGT 2995
Hoxa1 GAGCCTCCTCGGGAAAGCTT 2996
Hoxa1 GCAGAGGATTATTTCACTCA 2997
Hoxa1 GGGAGGGACAGATGACTGAG 2998
Hoxa1 GTGGATGGGACCCTTTCCAA 2999
Hoxa10 GAACTGTGGTTTGGGAGGTC 3000
Hoxa10 GCTGCCTCAAAGTGGAGGTT 3001
Hoxa10 GATGAGGAAGTCCATTCCCT 3002
Hoxa10 GACCAGCAATAGAAGCCTGA 3003
Hoxa10 GTGTGAGATCCAGACAGGGA 3004
Haxa10 GAGCGAGAGAGAAAGCAGTG 3005
Hoxa10 GATAGCACTCTGAGAGGGAG 3006
Hoxa10 GCAAAGAGTGAGAGGGCGAT 3007
Hoxa10 GCCAGATCTCTCATGCTGAA 3008
Hoxa10 GGAATGAGGGATTTGGGAGG 3009
Hoxa11 GAAGAAAGGGAGGTCTCTGA 3010
Hoxa11 GCTACTATTGAGCAGCCTTA 3011
Hoxa11 GCTTTGCCTGTTGGCGGTTT 3012
Hoxa11 GTGTGCTCTTATCCCTAGTT 3013
Hoxa11 GGCTGACAGAGCAATTCGAC 3014
Hoxa11 GAAGCCGCCTCTTCTAGAAA 3015
Hoxa11 GTGGGTGAGGGATACTCTCT 3016
Hoxa11 GAAAGGAAGCCGAGGAGGGA 3017
Hoxa11 GAGAGTATCCCTCACCCACC 3018
Hoxa11 GCTACAAAGAAAGGAAGCCG 3019
Hoxa13 GGGTCCCAGGACATTTCTCT 3020
Hoxa13 GTAGTGGGTTCAAGGTGCCG 3021
Hoxa13 GAATGCAACAGTGGATTGCC 3022
Hoxa13 GATGCAGCAGCTATTCTCTC 3023
Hoxa13 GGGCAAATCAATATTTACCC 3024
Hoxa13 GCGGTGTTTACAGGCTGGAC 3025
Hoxa13 GAACTGGTCAGACATCCAGA 3026
Hoxa13 GCTAGACCCTCCCAAGGATG 3027
Hoxa13 GCAGTAAGAAGGTAAACTCG 3028
Hoxa13 GAAAGGACTCCCTGGGTGTG 3029
Hoxa2 GAGGCAAGGAGGAAGCCAAA 3030
Hoxa2 GTTTCATACCCGTAGGGCTC 3031
Hoxa2 GTTCAAATGCTGATTATCTC 3032
Hoxa2 GAAGGTGCTTTGCAGATGGA 3033
Hoxa2 GATGGAAGGGTGGTGGCTTT 3034
Hoxa2 GAAGCTGAGATGTGTTCTTA 3035
Hoxa2 GACCTGCGTGTGGAGATTGG 3036
Hoxa2 GGAGGGTAGACGACGACGTG 3037
Hoxa2 GCTCCTAAACGCTGCTCTCT 3038
Hoxa2 GTGGGTAGAGGCCATGATGA 3039
Hoxa3 GTAGGAAAGACATGGAATTC 3040
Hoxa3 GATAAGAATGGAGACCTTCG 3041
Hoxa3 GGTATTGGCCGGGTGTGTGA 3042
Hoxa3 GGACATGAAGGAGGCTTCTT 3043
Haxa3 GCCAGAGAAAGAGGGATTCT 3044
Hoxa3 GAAACTGGCCCAGCCTAGTC 3045
Hoxa3 GGACGGGACATGAGGAGACA 3046
Hoxa3 GCATCAAGGTCCAGCCTGGG 3047
Hoxa3 GGCACTCCCAAACTACCTAT 3048
Hoxa3 GCCATTAACCCTACTTCAGG 3049
Hoxa4 GCAGTGCATGTGTATTTGTA 3050
Hoxa4 GACCGATTGACAATTAGACC 3051
Hoxa4 GAAGGCAAGAGATGCTTCTT 3052
Hoxa4 GGCTGTGGAAGGTTCAGGAA 3053
Hoxa4 GAGGTTCTATTAAGGAGGAT 3054
Hoxa4 GCTCTGGAAAGGAGAGAGAA 3055
Hoxa4 GTTCTGAAACGCGAAGTTAC 3056
Hoxa4 GCTCGCTTCTCCCACCCTGA 3057
Hoxa4 GCAGGGACTCCCTAACAGCC 3058
Hoxa5 GGGTCCTGAAAGCTGCGAGG 3059
Hoxa5 GGTGCCGTGTATGGGAGTCA 3060
Hoxa5 GGCTGCTTGGAAGCTGGGAT 3061
Hoxa5 GTCTGTGAAAGACGCTATCC 3062
Hoxa5 GCAGTGCCCTGTTTGGTGCC 3063
Hoxa5 GCGCGTTAGCGATCTCGATG 3064
Hoxa5 GGCTGCTACTCTCCCACTGA 3065
Hoxa5 GAGGACTGTGTTGGGCTGTC 3066
Hoxa5 GCCAGGTGTGAGGTTCAGGC 3067
Hoxa5 GGCACCTGTGGGCAGAAATG 3068
Hoxa6 GAGCCTGGCTTGCAGGTGTG 3069
Hoxa6 GCTTGTCAGGTTTCCTGTTT 3070
Hoxa6 GTCCTGACAGAGTGGAGACC 3071
Hoxa6 GCCGATGGTCAAGGTAATTC 3072
Hoxa6 GGAGGGCGGTACTGAGAAGA 3073
Hoxa6 GCGTCCCAAAGGCGTCCTGA 3074
Hoxa6 GAGATTTGACTGGATGGAGG 3075
Hoxa6 GATCCTTTGAGTGAAGCTCT 3076
Hoxa6 GCCTGTACAAACAGTCTCCA 3077
Hoxa6 GGAAGGCCCTGGCTTTGGTG 3078
Hoxa7 GCTTAGAAAGGTGAAGCCGC 3079
Hoxa7 GGGAACCACTTAGTCCTTTC 3080
Hoxa7 GAGACCTGACAACCAGAGTT 3081
Hoxa7 GGCTGTCTTGTGTAGATCTT 3082
Hoxa7 GACCCTAAGGCGGCAATATC 3083
Hoxa7 GAGTAAGAGAGAGAAAGAGA 3084
Hoxa7 GCTGCTGAGATTGGCGGAGG 3085
Hoxa7 GAGCCGCCAGGAGTGTATGA 3086
Hoxa7 GCCAACAGATATACTAACAT 3087
Hoxa7 GCAGTTTATGAGGCGTTTAG 3088
Hoxa9 GGTGGAGAGCCTAATATTTG 3089
Hoxa9 GTAGAGACCCAGCCAGAGAC 3090
Hoxa9 GTTAGGGTGGTGTCTCTGTC 3091
Hoxa9 GAAGGGTAAGCAACAAGGCC 3092
Hoxa9 GATCAGGGAGGGCACAAACT 3093
Hoxa9 GTCTCTGGCTGGGTCTCTAC 3094
Hoxa9 GTCCTGCCTTGTGCAACTGA 3095
Hoxa9 GGAGCCCTCTTCATCCACCA 3096
Hoxa9 GTGTCGTGCTGTCGAGAGAA 3097
Hoxb1 GAACCTATTGAAGGCCTTGG 3098
Hoxb1 GTGATCTCTCCCAGGCCAAT 3099
Hoxb1 GGTAACCCTTGAAACTTCTC 3100
Hoxb1 GCCTGAGCTAGGGCAAGTCC 3101
Hoxb1 GCGGAGGAAGCCAAAGCAGG 3102
Hoxb1 GATGAGTTGATGGATAGGTA 3103
Hoxb1 GATGCCGCATGGAAAGAGGA 3104
Hoxb1 GAGAGGCTGAGGGAGAGAAA 3105
Hoxb1 GGAGGGCAAGAGTTCAGGGA 3106
Hoxb1 GCCTCAAATACATAAATCCA 3107
Hoxb13 GGGAAATAGAGCCAATGTCT 3108
Hoxb13 GTCCCAAGATTGCAGGAGCT 3109
Hoxb13 GGTGAACAACAACCTGGATT 3110
Hoxb13 GAAGGGCTGGGAGGCCACTT 3111
Hoxb13 GGGAGCCAAGGCTGGITTCG 3112
Hoxb13 GGGAGCAAAGCAGGAATCCT 3113
Hoxb13 GCCAATCAGCGCTCATGCCC 3114
Hoxb13 GGGTCTGGATTTCCGTTTAA 3115
Hoxb13 GCTGCCTCAAAGGAGAACCC 3116
Hoxb13 GGCTGCCTCAAAGGAGAACC 3117
Hoxb3 GGCGACGCAGCTTTAAACAG 3118
Hoxb3 GAACCGAGATTGGAGTCATA 3119
Hoxb3 GTCCTGCGATGGTTTCGTTT 3120
Hoxb3 GTCTTCTGGTTTCATTCTAA 3121
Hoxb3 GGAACAGCGAGCACCGAAGG 3122
Hoxb3 GAGGCAACGTAGCTGCATCC 3123
Hoxb3 GCCAAGCATCCTAGAGGGTA 3124
Hoxb3 GAAGCAGAGAGGCCTCCCTA 3125
Hoxb3 GTTGCCTGTAGCCCTGGAGG 3126
Hoxb3 GCTGCATCCTGGGCCATGAC 3127
Hoxb4 GGGATAGAGAGATGCAAAGC 3128
Hoxb4 GAACAAGGACCCAAGCTTCC 3129
Hoxb4 GGAGAGGTGTCTGGGTGTGA 3130
Hoxb4 GCTCCCACCTGCAGGCAACT 3131
Hoxb4 GTCTTCTTGAAGGCAGTCAC 3132
Hoxb4 GGCCTTGTGGGTTAAAGGGA 3133
Hoxb4 GATCACAAACTAAAGGCTGT 3134
Hoxb4 GCAGTTCATTTCCGAATGAA 3135
Hoxb4 GACAGAGGCGGCGGCTTTAG 3136
Hoxb4 GAGCTCCAAGGGAGAGGAAT 3137
Hoxb5 GAGAGACACAACCAACGCTG 3138
Hoxb5 GCCAGAATCTATCATCGAGT 3139
Hoxb5 GCATCTGGCGAGCTTGTTAA 3140
Hoxb5 GTCTTTCAGGTCCCTGCTGA 3141
Hoxb5 GCGAAGGGAGAGGTCTGTGG 3142
Hoxb5 GACGCGAAGGGAGAGGTCTG 3143
Hoxb5 GGTAGTGTCTCACAGCTCCC 3144
Hoxb5 GTTGCACAGAGCCAGCAAAG 3145
Hoxb5 GTCTCAGCTCAGTGCGGAGG 3146
Hoxb5 GAGTCCAGGAGGGAATCTGG 3147
Hoxb6 GAGCAAGCATGCCAGTTTGA 3148
Hoxb6 GAAGCTGTCTTTGTGAACTG 3149
Hoxb6 GGGTTGCAGCGGTCAGTTCT 3150
Hoxb6 GAGGCCAGGCCAGCAAGTAG 3151
Hoxb6 GCTGCAAACCGCACAGGTGG 3152
Hoxb6 GTTGGATACACTGTTTGTCT 3153
Hoxb6 GAACCACCTCGGAGCTCTTA 3154
Hoxb6 GCACACACACACACAGGAGG 3155
Hoxb6 GGATTTATTTGGCTGCAATG 3156
Hoxb7 GTAGTAACTAGATGTGACCA 3157
Hoxb7 GGAAGGGAGGAAGGAGGCTT 3158
Hoxb7 GCAACTTGGTGGGTGGGTGC 3159
Hoxb7 GAGTCAGATAGGGATTAAAT 3160
Hoxb7 GGAGAAAGAGAAGCTGGAGC 3161
Hoxb7 GGGAAGAGATCTACCCAGGC 3162
Hoxb7 GCCGTCATACCATTGGCCGA 3163
Hoxb7 GAACTCCTTCTCCAGCTCCA 3164
Hoxb7 GGAGGAGAGAGGATCGAGGG 3165
Hoxb7 GAGGAGAGAGGATCGAGGGA 3166
Hoxb8 GGAAGCCGCAGCTCTCACCT 3167
Hoxh8 GGAATAAAGTGCAGGACAAT 3168
Hoxb8 GACAATGGGTCAGGTGAGAC 3169
Hoxb8 GGAAAGAGAAGAAGCCACAC 3170
Hoxb8 GGAAGCCAGTCCTTCTGGGA 3171
Hoxb8 GAAATAATAGGCACAAATCA 3172
Hoxb8 GATTCTCTCTTCAGCAGGTG 3173
Hpxh8 GTCATGATTTGAGGACTCAC 3174
Hoxb8 GCTGAAATGAGACCGATTAT 3175
Hoxb8 GCATAAcACAGCAGTAACCA 3176
Hoxb9 GACTGTGTGTGTGCTCTCGG 3177
Hoxb9 GGAGGCTAAGGAGGGAGTCA 3178
Hoxb9 GGCCCTGGAACTAGAGTTTC 3179
Hoxb9 GCAGCTGAGAGAGGCGAAAG 3180
Hoxb9 GGATGGAAAGGAAGGTAACC 3181
Hoxb9 GGAGCGAATGAATCATAGTT 3182
Hoxb9 GCAAAGCCCGGGAGAGGAAT 3183
Hoxb9 GCCCGACAGGGTAATTAAAG 3184
Haxb9 GGGAGGACCAGCATACAGGG 3185
Hoxb9 GTTAAGTATCTGTAGGTCCT 3186
Hoxc10 GCCAGGCAGGGACAATAGGA 3187
Hoxc10 GACTGTCCCAAGTCTGGTCT 3188
Hoxc10 GAGAGCGCTTGTGTGGGTCC 3189
Hoxc10 GCAGGAAGCATTTCTCCTGA 3190
Hoxc10 GGACTGTCCCAAGTCTGGTC 3191
Hoxcl0 GAAAGTGTAAGGTGAAGAGA 3192
Hoxc10 GGTCTAGCCGTCACATGGTG 3193
Hoxc10 GTGTTATTCAGGGCAAGGTT 3194
Hoxc10 GTGGAGTGT6TGGCCAGCAG 3195
Hoxc10 GAGTCTCCAGTGTCTGGAGT 3196
Hoxc11 GTAATAGCCAAAGGGACTGG 3197
Hoxc11 GGAAGTCTCTTCTACAATAT 3198
Hoxc11 GCACAGCCTTGGAGAGAGGT 3199
Haxc11 GCTAGACAAAGTTGGGACAC 3200
Hoxc11 GCAAGGAGGGTTTATAGACT 3201
Hoxc11 GGAGGAGAGAGAGAGAGGGT 3202
Hoxc11 GACTTGGAGAAGGGCAGGGT 3203
Hoxc11 GAGCACTTCGCAGACGTAGG 3204
Hoxc11 GCAACAGAATCTTCTGTTTC 3205
Hoxc11 GCTCTGATTCTTCAGGTAGA 3206
Hoxc12 GAACATCTGCAAGTCAACAT 3207
Hoxc12 GGCTAAGGGAGGGAACCAGG 3208
Hoxc12 GGTGATAAGATAATACATCT 3209
Hoxc12 GGAGATTAGCATTGTCGGAA 3210
Hoxc12 GCGTCCIGTAGAGGAGAGAG 3211
Hoxc12 GTGTTGCACAGAAGGAAGAG 3212
Hoxc12 GAGAAATCCACGTCTGAAGA 3213
Hoxc12 GGTTGCAGAGAGAATGAGAA 3214
Hoxc12 GAAGGAGAACCGGCCAAGCG 3215
Hoxc12 GAGATTACCCTACAACCTGC 3216
Hoxc13 GACTACCGAAGTCTCTAAAT 3217
Hoxc13 GTAATTACATCTCATTTCGG 3218
Hoxc13 GTAGCAGGCACGGAAGGTCT 3219
Hoxc13 GCTGCTGGAGTCCAAGGTCA 3220
Hoxc13 GGTCTAGGATTAGTCTTGAT 3221
Hoxc13 GCGTAGTGGGAATGCGGCTA 3222
Hoxc13 GGCTCCGGTTCTCAAACAGA 3223
Hoxc13 GTGATAAGCGCTAAGGAGCC 3224
Hoxc13 GCTGTGGTCACGTGGGAACC 3225
Hoxc13 GGGAGCTTGGCACAATTCCA 3226
Hoxc4 GACTTGAGGATCCGTGAATG 3227
Hoxc4 GAACTACAAGTTGCTGGAAG 3228
Hoxc4 GGGAAGGACAGTGGGTAGCA 3229
Hoxc4 GACAGGGTCCCAGCAGTACT 3230
Hoxc4 GGGCTTCAGTGCAGGTTGGA 3231
Hoxc4 GATGTCATTTCTGGAAGTCT 3232
Hoxc4 GTGTGTGGGTGACAGAGGGA 3233
Hoxc4 GGGAAAGCAGCCAGAGGCAC 3234
Hoxc4 GCCCACACAGGCTTCCCTTG 3235
Hoxc5 GCAGGAAGAAAGGCCCGCGT 3236
Hoxc5 GATGACTGAGAAAGAGAGTT 3237
Hoxc5 GAAGCTTGAGTGAGCCGGGT 3238
Hoxc5 GGGAGGTTAGTGATGGAAGC 3239
Hoxc5 GATGAGCAAGGGAGAAGAGA 3240
Hoxc5 GCCTTCTAGCAGTCAGTTTG 3241
Hoxc5 GGTCTCCTAGGCCTAGGCGA 3242
Hoxc5 GAAGTCTACCCAAGTTCACC 3243
Hoxc5 GAGACCTTGACCTTTAGTTT 3244
Hoxc5 GCTCAGAAGCCGAAGATCCC 3245
Hoxc6 GCTGTTGGAAACCTCTGCCC 3246
Hoxc6 GCATCCCGAAAGAGGAAATT 3247
Hoxc6 GAAATGGACTTTCTCCCTTT 3248
Hoxc6 GTTTCCCTGGAGTGTCACTA 3249
Hoxc6 GGACCCTCTTTCTACTGGGA 3250
Hoxc6 GCCTTACAACTCAGGTCCAG 3251
Hoxc6 GCAAGCCAGATGTCAAGAAA 3252
Hoxc6 GAAACATGGTGCACAGAGGA 3253
Hoxc8 GCTCTTTCCTCTAACAGCCC 3254
Hoxc8 GTCATCAAAGAAAGAATGGC 3255
Hoxc8 GGGTACATGATCACCATGCT 3256
Hoxc8 GCTGACATTTCTGGCCAGAG 3257
Hoxc8 GTGTGCTTCTAAGCCCAGGC 3258
Hoxc8 GTTTGCAGGTTAGGCAAGGA 3259
Hoxc8 GAGATGGGTCCTCACTCTAC 3260
Hoxc8 GGTGGCCTCACATACTGTAG 3261
Hoxc8 GAGGGTCCAGATCCTCTCTG 3262
Hoxc8 GCTCAGTACAGAACTGAACA 3263
Hoxc9 GTGACGTGAAGGCGGCAAAC 3264
Hoxc9 GGCGAGACATCTCAGAGATC 3265
Hoxc9 GCTTTGTGTGGGTCCTTGCT 3266
Hoxc9 GAAGTGGAGCAAGGTCTCAT 3267
Hoxc9 GTCTCAGACAGACAGGCAAG 3268
Hoxc9 GTCCAGAGCAGGTTGTCCGC 3269
Hoxc9 GGATTCTCTGAAACTCGGCA 3270
Hoxc9 GATGATGGATTTAAAGGAGG 3271
Hoxc9 GCACACAGCCAGTTTGGGTA 3272
Hoxc9 GGTGAAGGAAGATATGTATA 3273
Hoxd1 GAGCCCTGGACATCAGCTCC 3274
Hoxd1 GAATGAAATGACCAGAGGTT 3275
Hoxd1 GCTCCTGGGACAGGTATTGC 3276
Hoxd1 GTTTATAATCATCTGAGGAG 3277
Hoxd1 GGGCTCACTCCTGGACTATG 3278
Hoxd1 GGCTAAGTTGGCAGCAAGGC 3279
Hoxd1 GTGTGCTTAAGTATCTCCCA 3280
Hoxd1 GTCCACCTCACTAGCATAAT 3281
Hoxd1 GACCTTCTCAGAGGGAGGGC 3282
Hoxd1 GGTATTGCGGGAGAAAGGCA 3283
Hoxd10 GAAGGACGGCTCCCACACAC 3284
Hoxd10 GTGGAGGCTCTGGGCCTAAG 3285
Hoxd10 GGCCGGGAGAAATTCCTTTA 3286
Hoxd10 GGAGAGACGCTTTCGCGAAT 3287
Hoxd10 GGTGCTAATCAGTGGTTGTT 3288
Hoxd10 GTCTTCCGTTTCCTCTGGTG 3289
Hoxd10 GTCTCTGGGCCTGAAATCCA 3290
Hoxd10 GGGCAAGAAGGGAATAAAGA 3291
Hoxd10 GTGGTTGTTCTTTAATGAGC 3292
Hoxd10 GGGCTGGTTAATTTAGTACT 3293
Hoxd11 GAAGGGAGTGGTACTAAGCC 3294
Hoxd11 GAGATTGCTCAGGGCTTAGT 3295
Hoxd11 GATTTCTGTGGATCAGTAAA 3296
Hoxd11 GCCATGTCGTTGAACTTGAA 3297
Hoxd11 GAGAACCAACCGATCTCCCT 3298
Hoxd11 GATGTTGTGCATCTTGCTAA 3299
Hoxd11 GATAGGTGAGGCTGGAGCAG 3300
Hoxd11 GCCAGCAGACTTCACTTTAG 3301
Hoxd11 GGACTAGGTGTGAGAGTGTG 3302
Hoxd11 GAAGCATTTCTCTCTCTACG 3303
Hoxd12 GTTCAACTAACTTGCACATC 3304
Hoxd12 GAACAGCGTGAAGATTCCTT 3305
Hoxd12 GAGGGAAGGTGGGAGGAGGA 3306
Hoxd12 GGAATGAAGTGGGTCGATTA 3307
Hoxd12 GGGTCAGTTGCTACAACCTC 3308
Hoxd12 GCAGCCTGCGAAATAAGGGC 3309
Hoxd12 GAGCCAAAGCCTGTTGAGGG 3310
Hoxd12 GGTCCTGCTTTAGGCTAGCG 3311
Hoxdl2 GTGATGTGCTTCCCTTTCCA 3312
Hoxd13 GTGAGCTCTGATTTGAATCT 3313
Hoxd13 GTGGCTGCAAAGTCAACTCC 3314
Hoxd13 GGATGAGCTGTCTCGAATTT 3315
Hoxd13 GGGTGCGTGAGCCTCAAAGT 3316
Hoxd13 GGTTAGTCAAGAGTGCTGGG 3317
Hoxd13 GCTCTGATTTGAATCTAGGT 3318
Hoxd13 GACTGCTGAGGCTGATTATG 3319
Hoxd13 GGATTTGGAGTTCTACCTGT 3320
Hoxd13 GTGTAGGTTATGAGAGGTAC 3321
Hoxd13 GATTGCGCGACGGCCCATCT 3322
Hoxd3 GGGCTGCTTAGTTCTGGGTC 3323
Hoxd3 GAATTTACTGCAATTCCTTG 3324
Hoxd3 GTCCTCTTTGGAGATACCGC 3325
Hoxd3 GTTTCCAAATAAAGACCTTG 3326
Hoxd3 GAAGTCCGATGGGTTGAGTG 3327
Hoxd3 GGTTATCAGGATGCTCAAAC 3328
Hoxd3 GGTTAGAGGGACAGGAAAGG 3329
Hoxd3 GCCTTTCTGGAACAGGGCTA 3330
Hoxd3 GGCTCTGGGAAAGCAGAATG 3331
Hoxd3 GTGTCCATGGGRGAAAGGGC 3332
Hoxd4 GGCGGTGATGGTACTCACAG 3333
Hoxd4 GAGGCAATACCCAGTTTACT 3334
Hoxd4 GATTGAAATAAAGGCGGTGA 3335
Hoxd4 GGCTCTAGCTAAATGAGAAG 3336
Hoxd4 GGATTTATGCTTAAGTACAC 3337
Hoxd4 GTTCCTACTGGGAGTTGCAA 3338
Hoxd4 GGAGTTGCAATGGCAGCGGA 3339
Hoxd4 GCCTTCCTGCAGCATCTGTA 3340
Hoxd4 GTGTACTTAAGCATAAATCC 3341
Hoxd4 GAAGTGGGTTTGCAAATGGC 3342
Hoxd8 GGGTGGCATTTCCTAGGGCA 3343
Hoxd8 GTCCTCAAGATCAGAGAGCC 3344
Hoxd8 GGGAAGAAGGAATCATGCCG 3345
Hoxd8 GTGCTGAACCCTTTATCCTT 3346
Hoxd8 GAATAGGTCTGGGAATGGAA 3347
Hoxd8 GCCTCCGCCAGCCTTAAAGG 3348
Hoxd8 GGAGCCGGAGAGGAAACTGG 3349
Hoxd8 GTTTCCTCTCCGGCTCCAGG 3350
Hoxd8 GCAGATTTGCACAGGTGCCA 3351
Hoxd8 GGAGGCGAGAGAATGTGGGA 3352
Hoxd9 GTTCCTTCTGCTTTCTGAAA 3353
Hoxd9 GGGAAAGAGAAAGGGACTTG 3354
Hoxd9 GGAGATCGCAGGACCCAGAG 3355
Hoxd9 GTTATGAAGACTGAGCTCTC 3356
Hoxd9 GTTGCTTGTTCCTGCAATGG 3357
Hoxd9 GGAACCGCATCTCCGAGGGT 3358
Hoxd9 GGAGCCGACAGTGATGGCCA 3359
Hoxd9 GTTCTTACTTACCAGGCTCA 3360
Hoxd9 GCAGTGGGTTCTTACTTACC 3361
Hoxd9 GGTTCCTGCAGGCCTCTCTG 3362
Hsbp1 GAAGGCGGAGCTAAGAACTG 3363
Hsbp1 GTTTAGTAAAGGAGAGAAAC 3364
Hsbp1 GAATTGTACAGGCACATGGA 3365
Hsbp1 GGAACATGGAGAACTCTGGC 3366
Hsbp1 GCGCCGAGAAACCGAAGTGT 3367
Hsbp1 GTGTCCTAGGAAGGAAAGAG 3368
Hsbp1 GTAATTCCCATCTCTGTGTT 3369
Hsbp2 GGCGTAGCTTATCCGCAGGC 3370
Hsf1 GCTTTCCGACTTGTCCGTCA 3371
Hsf1 GGAGCTCAAATGTGTGACGA 3372
Hsf1 GTGTCAGTTTCAGGACCCTT 3373
Hsf1 GAAAGAGGAAGTGCCTGCCT 3374
Hsf1 GCAGCGATGCCGGTGACATG 3375
Hsf1 GGGTGAGGGACCAGCTTCCA 3376
Hsf1 GGCTCTCCTGCACATGAGGA 3377
Hsf1 GTCCACAGAGGCAGGAGAGG 3378
Hsf1 GGTCTTGCCAAGTGCCTGCA 3379
Hsfl GCTCTGATTGGTGAGCAGCC 3380
Hsf2 GTGCATCCGAAGCCTTGGAT 3381
Hsf2 GCAGAGTGCAGAGAAGCCCA 3382
Hsf2 GCGAGTCCAATCCAAGGCTT 3383
Hsf2 GGATTGGACTCGCTGAGACC 3384
Hsf2 GCACTACAGAGCAAAGCGAT 3385
Hsf2 GGATTCGCATGGAAAGGGTT 3386
Hsf2 GTCTCAGCGAGTCCAATCCA 3387
Hsf2 GATTCGCATGGAAAGGGTTT 3388
Hsf4 GAATTTATGGTGCCTGAGAT 3389
Hsf4 GCAGGTCGAGGTGGCGAAGT 3390
Hsf4 GACTCGAGATCACAGGACGC 3391
Hsf4 GGAAATACACAAAGGCTGGA 3392
Hsf4 GACATTCAAGGAGACCTCAA 3393
Hsf4 GCCTTTGTACTGTGACTGTG 3394
Hsf4 GACACATTGAAGCCTAGGTA 3395
Hsf4 GGCCTTTGTACTGTGACTGT 3396
Hsf4 GGGCCTTTGTACTGTGACTG 3397
Hsp90ab1 GAAGGTCTCTGTGAATAATG 3398
Hsp90ab1 GCTGACCTGGATCGGTCACA 3399
Hsp90ab1 GGTCGTTCAACCCTGGCCCA 3400
Hsp90ab1 GATCTGCTACCATGACGTCA 3401
Hsp90ab1 GCAGGCCACCTTTAGAACAG 3402
Hsp90ab1 GGACTGAAAGAGAATGGAGG 3403
Hsp90ab1 GGAGCATGCATATGCAATTA 3404
Hsp90ab1 GAGAGGCTAACAGACAGTGC 3405
Hsp90ab1 GGCCTCCTTAAAGTTGGACA 3406
Hsp90ab1 GTACAGCACAGCTTCAGGCT 3407
Id1 GAGTGTGAGGAGCTGAGGAG 3408
Id1 GACGCTGACACAGACCAGCC 3409
Id1 GAACGTTCTGAACCCGCCCT 3410
Id1 GGTCTCTTTCTCACTTCTCC 3411
Id1 GCGAAGGAATCCAACTCAGC 3412
Id1 GGCTCAAGAACTGAAAGGGT 3413
Id1 GCATAGGTAGAGCAGCTAGT 3414
Id1 GATCCAGAGGTGGGACCCAG 3415
Id1 GGCTCAGACCGTTAGACGCC 3416
Id1 GACCAGCCCGGGAAAGGAAA 3417
Id2 GACGTGCCCAGCTGCAGTAA 3418
Id2 GTCTTAAGTTTCGAGTGATT 3419
Id2 GTAACCCTGCCTCATTCTTG 3420
Id2 GGGTGGTGGGAAGGTGTGAA 3421
Id2 GGGCATCCCTGAATTGCCAT 3422
Id2 GCAGCCAATGCCTGTAGGGT 3423
Id2 GCCTCCTTAGAGAGAAGCCC 3424
Id2 GATCCCGCCCTTACTGCAGC 3425
Id2 GCCTCATTACCCCAACAGAA 3426
Id2 GCTCATTATCATCCAGCCCA 3427
Id3 GCTGATACCGAGGAGAGGCG 3428
Id3 GCTGATACCGAGGAGAGGCG 3429
Id3 GCTTTCTTAATCAATCAGCC 3430
Id3 GCTTACCTGTGATGTGATAC 3431
Id3 GGCGTGGAAAGGACTGAATG 3432
Id3 GTGCAAAGTGTGCAAAGGGA 4433
Id3 GTTCTATGTATGCCCGTGGA 3434
Id3 GCAGGCAAGAGAGAGTCTTT 3435
Id3 GAACGCATGACGTCCCACCC 3436
Id3 GAGTGTGCAAAGTGTGCAAA 3437
Id4 GTTACATCCATCTATGAAAC 3438
Id4 GGGAATGACGCTCGGGCCAA 3439
Id4 GGGACTCTGAGCCTTGTTTG 3440
Id4 GGTGGCACTGTCCTCCTGAT 3441
Id4 GAGCTTAAAGGTAGCAGTAT 3442
Id4 GCCTTCTCTATAGACAGCGT 3443
Id4 GAGGAGCATGAAGCCCATCC 3444
Id4 GCCTTCTGACCCTCCAAAGG 3445
Id4 GCCTCTAAGGATTTAGAGGG 3446
Id4 GACGCTCGGGCCAATGGGAA 3447
Ikzf1 GGAAGGCTCTGGGCCTCAAA 3448
Ikzf1 GTGCACACGCCAGCGTGGAA 3449
Ikzf1 GCTAGACTGGTGGTAAGGAA 3450
Ikzf1 GCTCAGGGTTAACGCCTGTC 3451
Ikzf1 GAGGAGTGAGCCACAGGGAC 3452
Ikzf1 GCGCCAACCCAAAGTTTGCA 3453
Ikzf1 GGCTGCAAAGTTTGTGTGCG 3454
Ikzf1 GGGCTTTCTGATATCATCTT 3455
Ikzf1 GCTGGTGGAAAGGAAGACAC 3456
Ikzf2 GTTGGCCTAGGTTCCTTGCT 3457
Ikzf2 GGCAGTGGATCTGTAGCTAA 3458
Ikzf2 GGTTATCCAATCTTTCTTCT 3459
Ikzf2 GGGAAGTTCTCTCTCTGGCC 3460
Ikzf2 GCTCCGCCGGATCGGTTTCT 3461
Ikzf2 GCCACATCCACCCGAGTCAA 3462
Ikzf2 GTCGATTCTTAAGGAACCGG 3463
Ikzf2 GCAGTGCACAAACACACGTT 3464
Ikzf2 GGTTTCTCAGAAATGTTGTT 3465
Ikzf2 GAGGATCTGGGACACTGAGC 3466
Ikzf3 GTCAGATGACAAGGTTTGTC 3467
Ikzf3 GCACTAGTTAATGTGAGCTC 3468
Ikzf3 GCATTTCTAACGATGCCAAC 3469
Ikzf3 GAGGAACTGTACACTTTCAC 3470
Ikzf3 GGGTAGAGGGACTAAGTCAA 3471
Ikzf3 GTTCCAGGTCCTTCCGTGTC 3472
Ikzf3 GAAAGCATTTGACAGGAGGG 3473
Ikzf3 GACGTCTACTTGAGAAACAC 3474
Il6 GAGAAAGCCTCTTCCAGATG 3475
Il6 GAAAGCACACGGCAGGGAAT 3476
Il6 GCAGAGAATAGGCTTGGACT 3477
Il6 GAACTCTTGTCAGCCTCATC 3478
Il6 GAAGCCCTGGTCTTCACAAA 3479
Il6 GAGAATGCAGAGAATAGGCT 3480
Il6 GTGAAGACCAGGGCTTCACA 3481
Il6 GTAACCCAGTGTAAACACAC 3482
Il6 GGTCATGCAAGGAGGCTTGA 3483
Il6 GTCTGTAGCTCATTCTGCTC 3484
Ilf2 GTGAATGGTAAGCTCTTCTA 3485
Ilf2 GGAGTAGATCCTAAGGACCC 3486
Ilf2 GGCTTCTTTCACTTGTCCCA 3487
Ilf2 GACTCACCTGGACTTGGCTG 3488
Ilf2 GAACAGGTAGAAGACTCACC 3489
Ilf2 GAGGGAATAGTGGTGGTAAG 3490
Ilf2 GCCAATAAGAAGACATAACA 3491
Ilf2 GTGAGTCTTCTACCTGTTCC 3492
Ilf3 GGTGGATCGCCCACGTGATG 3493
Ilf3 GGTCCGGAGCTCTTCATATC 3494
Ilf3 GGGAACACCGGCAAGGTAAG 3495
Ilf3 GCACATGCGGTTGTCAACAC 3496
Ilf3 GCCAAGAGGACGCTAGTGAC 3497
Ilf3 GTGGATCGCCCACGTGATGC 3498
Ilf3 GATGGAAGAGCCGAGCGAAG 3499
Ilf3 GATATGAAGAGCTCCGGACC 3500
Ilf3 GACCTGAGGTGCTTCTGATC 3501
Insm1 GCCTGTGGAGTTGACCCAAG 3502
Insm1 GGCTCCCTCTGAGGACAGAT 3503
Insm1 GGTGTCCACTTGGGACACTT 3504
Insm1 GGACCGCAGCTGCATCCATA 3505
Insm1 GGGTGAGCTTTGCTCTTCTC 3506
Insm1 GAAGGAGGAGACCCACAGGT 3507
Insm1 GACAGGGACGCGTCCATGAA 3508
Insm1 GTACATCTGCCGCACCTACC 3509
Insm1 GAGCAGCAGACCGTGAAGGG 3510
Irf1 GTTCTAGCTAGCGGTGACCA 3511
Irf1 GAGCGATTCGCAGAGGGTGC 3512
Irf1 GACGAAGGAGTGGTGCGCAC 3513
Irf1 GCTGGGAGTCTGCAGAAAGA 3514
Irf1 GCTTTCAGTAGGGTCTCTGT 3515
Irf1 GCACGGGACACCAGGAAGTG 3516
Irf1 GCGAAAGATGCCCGAGATGC 3517
Irf1 GAGACACTCTGACCAGCCAA 3518
Irf2 GCAGGTGTAACTAAATGTAA 3519
Irf2 GAACTTCCGCACCTCCAGGC 3520
Irf2 GTTCTGAGCACTTAAGCCAC 3521
Irf2 GTCTTCTCTTCCCTAGAACA 3522
Irf2 GATTCCACAGACAGGGATAA 3523
Irf2 GTGGTGGCCGTAGGGAAGGA 3524
Irf2 GCCTTTCCTCTCCCTGTTCT 3525
Irf2 GCTAGACCGTGTGGGAAAGA 3526
Irf2 GCAGGAACGTTGTTAGTTCC 3527
Irf3 GAACTCACCTGGGTGGAGTT 3528
Irf3 GGTGCTTGGAAGTCACAGCT 3529
Irf3 GGTGTGACAAAGACTTGAAA 3530
Irf3 GGTCACTTGTGAAACTTTCA 3531
Irf3 GTCAGATTACCAACTGGCCA 3532
Irf3 GAAGAGGGCGCCTAACTCCA 3533
Irf3 GATCTTCCATGAAAGGATGA 3534
Irf3 GTTCCCAGCATGCCTGTAGG 3535
Irf3 GAGCAATTCCGTGGTTGACC 3536
Irf3 GAGACCCAACTCTTCAGAGC 3537
Irf4 GTGGTTGTCAGGGCTCACAG 3538
Irf4 GAAGTGATAGTTTAACAGTG 3539
Irf4 GTTGCCATGATTGAAACTTT 3540
Irf4 GAAACAAGGTCTCCGTCTCT 3541
Irf4 GGCAGACTGGTTAAAGACAT 3542
Irf4 GAACTTTATAGACCGGGAGG 3543
Irf4 GTTCTTAGTGGTCAGCTAGA 3544
Irf4 GGAGGAGCTGAAGAAAGCCA 3545
Irf4 GCTTCGGACTAGAGCCCACC 3546
Irf4 GGTATGCTGTTTGCAAGGAA 3547
Irf5 GCAATTGTGAACTGGCAGGC 3548
Irf5 GGCAGGAGGGAGCTTCTGTG 3549
Irf5 GATCTCTGAGTTGTCCCATC 3550
Irf5 GCTTTGCAGGTTCTCTGGAC 3551
Irf5 GAAGGCCCGTTTATGGAACC 3552
Irf5 GAGCTGTGTGCCGACAGGGT 3553
Irf5 GTCGATGGAGCCACACTCCA 3554
Irf5 GTTGCCTTGAACTGGGTGTG 3555
Irf5 GCTAGCTAAAGTGAACAATG 3556
Irf5 GGACCAGAAAGGATGTGGAC 3557
Irf6 GTGACATCCCAAACTGAGCT 3558
Irf6 GTGCAGGGTCACTACGGGAG 3559
Irf6 GGACATTTGCTTGGTTTCAA 3560
Irf6 GGGAAAGCTCAGGTCTTCCC 3561
Irf6 GATGTCACCGGGCAAAGGCT 3562
Irf6 GTGGCACTTGTCAGGCACAC 3563
Irf6 GTGTTGTGATCGACTGAGGG 3564
Irf6 GTCCACCACTCAGGAGACTG 3565
Irf6 GAAGGGTTTGCCTCACTGCC 3566
Irf7 GGCTGCTTTGGCAATGAACA 3567
Irf7 GAATTCCAGAGTCTTAAGGC 3568
Irf7 GCTTTCCTCTTAGCTACAGT 3569
Irf7 GAACGTGCGTGTGGAGTGGA 3570
Irf7 GACAGCTTCACGTGAGGGAG 3571
Irf7 GTGGGTAGACCTTTAGGGAA 3572
Irf7 GCCAGTGCCTCGGGAAGTGA 3573
Irf7 GCATGCCATGACTGCTGTTC 3574
Irf7 GTGTGTAACTACCGTAGCCC 3575
Irf7 GGTGTTTGGGACCCTCATGA 3576
Irf8 GAGCACCGATTCTCCTCAGA 3577
Irf8 GCGCGAGCTAATTGAGGAGC 3578
Irf8 GGGAGAGGTGTTTGTTCATT 3579
Irf8 GCAGGAAATCTGGGAAACCA 3580
Irf8 GCATGTGCAGGGCTTAACTA 3581
Irf8 GGAAACCCTGACCTCAGCAG 3582
Irf8 GCCTGAGCAGCTGACACTCA 3583
Irf8 GGCCTCTAAGGATGAGGGTG 3584
Irf8 GTGGCCCAGGGCTGAATGAA 3585
Irf9 GAAGGACCACCAAGAAGCCT 3586
Irf9 GTGAACATATGCAAGATGGA 3587
Irf9 GCAGTAAGCTGAGGTCTCTG 3588
Irf9 GGCAGTAAGCTGAGGTCTCT 3589
Irf9 GGCAACACGGCTTAGTCATT 3590
Irf9 GGAGAATTGAAACTTAGGGT 3591
Irf9 GAGAATTGAAACTTAGGGTG 3592
Irf9 GATGGGCAATAGCTCCCTGC 3593
Irf9 GCCATAAGATGCCTCTTTAT 3594
Irf9 GGGTTCAGGGATGAAGCTTG 3595
Irx1 GTCGTGGGAGACTCAAAGAC 3596
Irx1 GGGATTGCGTTTCTACAGCT 3597
Irx1 GTGGACTCCCTGGTCAGGTC 3598
Irx1 GCAAGGGTGTGGACTCAGTT 3599
Irx1 GGCCTGGCTGCTCTGTTCTC 3600
Irx1 GAGGAGAGCTCCTAGAGGGT 3601
Irx1 GCTGAGAGGTCGCTGCCTAG 3602
Irx1 GTTTACAGCTGTCTGACACC 3603
Irx1 GCAAGCAAGTGTGCCTTAGC 3604
Irx1 GTTGTGGGAGTAATGACAAG 3605
Irx2 GGACTCTGAAACCTGGCGCG 3606
Irx2 GGGTAGATGCTTGGCAGCCC 3607
Irx2 GCTTCAAGGAGACACCTGTT 3608
Irx2 GCTCTACCTAGCAAGCTTCA 3609
Irx2 GTTGGAAACCAAGAGCAGTT 3610
Irx2 GTCACGGATCTGGCTGCTGC 3611
Irx2 GCTCTGAAGCTAGTAGAGGG 3612
Irx2 GTCCAGGTCCCAGGGAACCA 3613
Irx2 GCTATCTTCAGGGTGATGAG 3614
Irx2 GCCTCAGCCGOGAGTACAGG 3615
Irx3 GAAAGTCATACTGAAATTCC 3616
Irx3 GAACGTGCTGCCTGGGAGTT 3617
Irx3 GGGTTCGATGTCAGCATGTG 3618
Irx3 GGTGCATCGGGAGTTGATTG 3619
Irx3 GGTTGGCTTTAAGGTGAGCC 3620
Irx3 GTGCCTGTTGGGAGAAAGAG 3621
Irx3 GGCGACAGAGCCAGATTGCA 3622
Irx3 GCTTTGTGCACTGTGCCTGT 3623
Irx3 GGAATGGATTTCTTTCTCCC 3624
Irx3 GCTACTTATCAGAACTTTGC 3625
Irx4 GAAGCTAAGGCTCACGGGAG 3626
Irx4 GACTCATTTCATGCTCACCG 3627
Irx4 GCTCTGGAGCCTTCCATGGG 3628
Irx4 GGGAAGCTGCCTTGCACAGT 3629
Irx4 GGAGTCTTCAAGGGAGCGAA 3630
Irx4 GCAAAGTCCAGGTGAGGAGG 3631
Irx4 GATGGTCCGGAAGGGAGAAG 3632
Irx4 GCCCTCACAATGCTATCCTT 3633
Irx4 GCCTGGGTCTTTGTAATCTG 3634
Irx4 GAGTAAGCTCCCGCCCAGAA 3635
Irx5 GGCAAAGCTTGATCATTAGC 3636
Irx5 GGGATGTGATTGTCATCCTG 3637
Irx5 GGGCCGTTCGGGAACACAAA 3638
Irx5 GATTACGTCATCCAGAGGAG 3639
Irx5 GCAAGCAGTTTGCTCGGTTG 3640
Irx5 GCCTCTTCTCGTCGTCTGCC 3641
Irx5 GAAGCCACTGTGGAGCGTGG 3642
Irx5 GTGTTCCGAGAACTCTGCCT 3643
Irx5 GCCTCTCGGGCTGACCATTC 3644
Irx5 GTCAGGTCTGTGGAGCCGGA 3645
Irx6 GGCTGCACCTCGATCTGGAG 3646
Irx6 GGCCAGGTCCTTGACCTCTT 3647
Irx6 GCGTTCTCTGTGGTCCAAAC 3648
Irx6 GATTCATTAAGTTAGTCCCT 3649
Irx6 GAAGGAGTTTATTACACCAT 3650
Irx6 GACAGGGCGACTGAAAGGTA 3651
Irx6 GTCCCGGGAGCTCTTAGGGT 3652
Irx6 GCTGCACCTCGATCTGGAGG 3653
Irx6 GATTCTAACAACCAAGCGCC 3654
Isl1 GAACTCAGTAATAGTAGGAT 3655
Isl1 GTAGCATGCCCTTGTACGGA 3656
Isl1 GTAGGTCCTTCCTGTGAAGC 3657
Isl1 GCTTTCTAATTTGTTTCCTC 3658
Isl1 GTCAACCTGGCTCTATAGAA 3659
Isl1 GAATCTTATATAGGTGAGGG 3660
Isl1 GCTCTCTGACATCCTATGTG 3661
Isl1 GTTCCTCCTGAGCTCCCTGC 3662
Isl1 GGGAGGAAAGGAACCAACCT 3663
Isl1 GGGAACTGCTTCTCTGGGCT 3664
Isl2 GGTGGGCCTGAGCCTTTGTT 3665
Isl2 GTCCAGTGCTGGCATGAGAG 3666
Isl2 GCCCGAGATCTATCTAATTC 3667
Isl2 GGAAAGCCCTGGAGAAAGCC 3668
Isl2 GTAATTTACCGTCTTCCCGG 3669
Isl2 GAGAGGAGAAAGGAGAGGGT 3670
Isl2 GAGATTGGCTGGGAGGAAGT 3671
Isl2 GGCTTTCTCTAGGTAGGAGA 3672
Isl2 GACGAGCTCTCTGTCATACA 3673
Isl2 GCTCCCAGCAAGGGCAAGAA 3674
Isx GGAGTGACAGGAGGAATTTA 3675
Isx GTGTAACCAAGGAGGAGGGT 3676
Isx GAGGTTTATAGGGTGAACCT 3677
Isx GGTGTTGGGTGGAGAGCTGA 3678
Isx GCTGTCTTGAAGACAGTAAA 3679
Isx GCTCCAAGCCCAGAGTTTAC 3680
Isx GAGCCTCACCCATCACACCC 3681
Isx GTCTTACTAGTACAAATCCA 3682
Isx GAAACCGAGGCTCAGAACAA 3683
Jun GAGAATAAAGTGTTGTGCCG 3684
Jun GTTTGGCTGTCTAGTGACGG 3685
Jun GATATGACTCCACCAGTGAG 3686
Jun GTAAGTGCGTTGAAGTTGAG 3687
Jun GAGGAACTCGGTTTCCATTT 3688
Jun GGGCTGCGGAGAGAGGAACT 3689
Jun GAGGTTTGCTTGGCGAGGGA 3690
Jun GCTGGAAGCTCAGTTGGGAA 3691
Jun GCAAGCCAATGGGAAAGCCT 3692
Jun GCAAATCAGGGAGGGAGGAA 3693
Junb GTGTCTGCAGGAGACTAACC 3694
Junb GGACTGTTCCATTGGCCGGC 3695
Junb GGTAATCGGAGTAGAAAGAT 3696
Junb GCTAGGCCAAAGCCAAGTCC 3697
Junb GAAGAGAAGAGTGGGAGGCT 3698
Junb GGTCTCTGGTAAGATAAAGG 3699
Junb GGTGAGTCAGTGTGGTCTCC 3700
Junb GGAAAGGGCCAAGACACAAC 3701
Junb GGTGACTAAGGGAGGGCTTT 3702
Junb GTAAACAGCGGCCACGAGCC 3703
Jund GGAGCCTGCAAATGAGAATC 3704
Jund GGCAACAACTGGTCAAGGCT 3705
Jund GCGAAGGTCCTGAGGTGCAA 3706
Jund GCTCCTGCTGATGGAAGTTC 3707
Jund GGTGCAAAGGAGCTCCAATG 3708
Jund GAGTGTGAGGGCAGAGCTTG 3709
Jund GCTGGGAACGGAGGTGGAAG 3710
Jund GGATCTCTCACCTTCCAGCT 3711
Jund GCATACTGTACTAATTAAGA 3712
Jund GCAACCAAGTTTCCAAATAA 3713
Kat2a GCTGTTGGTAGTTCTGATGG 3714
Kat2a GTGTCTGAAGTGACCTGTGA 3715
Kat2a GACCATCAGCTGATTCTGAA 3716
Kat2a GGCCAGAATCTGACGGTGAC 3717
Kat2a GCCCTTCTGGATGGGAAGAG 3718
Kat2a GTGACAATTACTATCCTCTT 3719
Kat2a GCCTGATCAGCTGCCAGAGA 3720
Kat2a GCCTGCCCTATTGTGGCTGC 3721
Kat2b GGGTGGTATGCTTATGCTCT 3722
Kat2b GGAAGACCAAGAATGAGCAA 3723
Ktt2b GTTTGCAGAGTGAATGCTGA 3724
Kat2b GCATTCACTCTGCAAACATT 3725
Kat2b GTGGAAGTAAACAGGAGTGA 3726
Kat2b GAGTCATCTCCCTCCCTCTC 3727
Kat2b GAACCGAATATGACCAGAGA 3728
Kat2b GTATCTCATGGAAGAATTCC 3729
Kat2b GAGGAGGATTGGCCGCTGAC 3730
Kin GTTGTATATTAGAATGCCCA 3731
Kin GGCGCTCCAGCACTGAACTA 3732
Kin GGGAGCAGCGTGACCCTTTA 3733
Kin GCCTTGAAGGTCGGGCAGAC 3734
Kin GGTGCTAGAGACTCACCTCA 3735
Kin GTGAACCTCTCGAGCCTTTA 3736
Kin GTTTCTGTACCAGCCTCCAG 3737
Kin GTGTTCACTAGTTAGCTAGT 3738
Klf1 GCCTAACGGCTCATTGTGTG 3739
Klf1 GACCCTCACTGGCTACTGCA 3740
Klf1 GTGCCCTATGAGTCAGGGTA 3741
Klf1 GGGTGCTGGTGGTTGTCTAG 3742
Klf1 GGCTACTGCAIGGAGCTGAA 3743
Klf1 GGTGCCCTATGAGTCAGGGT 3744
Klf1 GATAATGCCTGAAAGGGAGC 3745
Klf1 GGGTGTGTGCGATATGTGTG 3746
Klf1 GTCTGGGTGGCTAAATAGAC 3747
Klf10 GAGTGATCACAGCAGGAAAG 3748
Klf10 GGGTAGGAGAAACTGGGTAG 3749
Klf10 GAAGGACAGTGCTTATTGAA 3750
Klf10 GTACCAAAGAGCTAGTGGCG 3751
Klf10 GCGGTTTCTTGGTAGGGCGT 3752
Klf10 GGAGAACCAGGGCGAGATGG 3753
Klf10 GGAGGACTGAAGGCTAGGGT 3754
Klf10 GGGCGGAGGACTGAAGGCTA 3755
Klf12 GATTTGACCATCTCTTGCCG 3756
Klf12 GAGTCACATTGATCCTGCAA 3757
Klf12 GGCTGTATAGCTCTTCACCA 3758
Klf12 GAAAGTTGCAGGTCATGTTA 3759
Klf12 GCAATCAGCTCTAACTTCTT 3760
Klf12 GGAGTAGGGAAATGCAAGCC 3761
Klf12 GCTGTATAGCTCTTCACCAA 3762
Klf12 GGGAAGCCACCTGACGATGG 3763
Klf13 GAGAGGGTTCTACTGGCCGC 3764
Klf13 GGATATTTCTATCTGGGTTT 3765
Klf13 GGTGAGGAGGTGGCTGGAGA 3766
Klf13 GTGTGTTAAGATTGGTTCAA 3767
Klf13 GTTTGAAGCCTCCAGGACCG 3768
Klf13 GCCACAGACAGTCATCTCAT 3769
Klf13 GATAGAGACAGTCTCTCCTC 3770
Klf13 GGTGGCGTATCGGTCCCTAT 3771
Klf13 GTCTAGACTTTAGAGCAAGG 3772
K1f13 GCCACCAGAGAACTCCGCTG 3773
Klf16 GGTGCTCTGCTGGTACACGA 3774
Klf16 GGTGGCAGAGGTCCTTGCTC 3775
Klf16 GTGCTCTGCTGGTACACGAG 3776
Klf16 GAAGCTGAACCAGGCTTCAT 3777
Klf16 GGGCTGCTACATGCAATGGC 3778
Klf16 GGAACCCAAAGTTCTCAACG 3779
Klf16 GGAGCGATTGGAAACTTCCA 3780
Klf16 GACCTCTGCACAAATCTAGC 3781
Klf16 GGGACCATCATTTCACAACT 3782
Klf16 GCCTTGGGAGGTGACGATCC 3783
Klf2 GAGGAAGTATGTGGTGAGCC 3784
Klf2 GCTGGCTCAGTGCTTAAGAG 3785
Klf2 GAGGGTAATAGAGAGAGGGA 3786
Klf2 GCACTAGAAGGATTTATGTG 3787
Klf2 GTATGTTTGTGGGAGGTGAA 3788
Klf2 GCAAGAGGGTAATAGAGAGA 3789
Klf2 GCGGTATATAAGCCTGGCGG 3790
Klf2 GGCGACGGCGTCAACAAACC 3791
Klf2 GACGGAAACGCGTCCCGGAT 3792
Klf3 GTTTCTTGGGTGACTCAGTT 3793
K1f3 GACGTAGGGACAGGGCATCC 3794
Klf3 GTGGCCTCACGCAGCCTTTC 3795
Klf3 GACCTTTCTATCTGTACCGA 3796
Klf3 GCCTGGGCTGTTTAGGAAGC 3797
Klf3 GATGCCCTGTCCCTACGTCG 3798
Klf3 GAATGAATGGTAAGAGGGTA 3799
Klf3 GAGGATTTAGATAAGCCGGA 3800
Klf3 GAACAGGACATTCGTCATGA 3801
Klf3 GCTTGGTGCTAGGCTAGAGT 3802
Klf4 GGATGACTGCCCAGCTGTGG 3803
Klf4 GGCGTTCCAGATTTACATTG 3804
Klf4 GAAAGGGATGAGTTGTGAGC 3805
Klf4 GGGACCCTAGTGCTCCAAAG 3806
Klf4 GGCAGTAGCCAGAGCTAGGG 3807
Klf4 GTGCGTATGCGAGAGAGGGC 3808
Klf4 GCAGTTGGCAGATGATGTAA 3809
Klf4 GATAATGGAAGGAACAAGGA 3810
Klf4 GAATCTCAGAAGCTAGGAGA 3811
Klf4 GACAAGCGCGTACGCGAGCA 3812
Klf5 GTGCTCAAATAACTCTGAGA 3813
Klf5 GTCAACAGCGGTGTTTGTCT 3814
Klf5 GTAATTTCTGGCATAGAGAT 3815
Klf5 GGGCTGCAATCCTCTTTCTG 3816
Klf5 GTGAATGTTTGTGCCTTCTT 3817
Klf5 GCGGGTGGAATCTAGGAAGA 3818
Klf5 GTGCAGCCAGCCAGTGTGAA 3819
Klf5 GAGAGGAGCGGGTGGAATCT 3820
Klf5 GGCTAGGAGGGTAAGCATAG 3821
Klf5 GGAAGGTGAGTGGTTTGGTT 3822
Klf7 GTCCTCTCCGAGTGCCGCAT 3823
Klf7 GCAAATGGCAGTAAAGGCCT 3824
Klf7 GTTTGGTGGAACCCACACTC 3825
Klf7 GTGACCATGTAAGGTAAACA 3826
Klf7 GTACCCAGTGCAGATCCGAG 3827
Klf7 GTAACTTCATGGAGCAGGTA 3828
Klf7 GACCATGTAAGGTAAACAGG 3829
Klf7 GGCACTCGGAGAGGACCATG 3830
Klf7 GACCATGAATTTGAGAGGGA 3831
Klf7 GATCTCCCTGCTGCCTTACA 3832
Klf9 GAGAGGTGCGTCTAGAACTA 3833
Klf9 GAAGGGCCCTTCTGACTGGC 3834
Klf9 GGATGGGCGTAACTGCCTAG 3835
Klf9 GGGCCTGGATTGTGACGTGA 3836
Klf9 GGGATGGGCGTAACTGCCTA 3837
Klf9 GGCTCCTCTAAAGCAGAGTT 3838
Klf9 GCACTCCTCCCTGTTCCTGC 3839
Klf9 GGCTACCAAAGATTAAGGGC 3840
Lbx1 GGCAGAAATGCTGATAGTAG 3841
Lbx1 GATCCTCCCTATAGGCAGAG 3842
Lbx1 GTGTTATAACAGGGAAGGGC 3843
Lbx1 GCAAGATTGCAGAAGGAGGT 3844
ibx1 GGGAGTGGGACAGAAAGAAT 3845
Lbx1 GGAAGGAACAAGAGGGAGAA 3846
Lbx1 GAGATTGGGAGGTGGGAGGC 3847
Lbx1 GTACCCTGTGCCCTCCTTCA 3848
Lbx1 GAACAGGCTTCTTCGGTGCA 3849
Lbx1 GTAAGAACTGGAGCCCAGGC 3850
Lbx2 GGCCTCAGAATCAGAGGGAA 3851
Lbx2 GCATATTAAGTGAAACCACA 3852
Lbx2 GAGTCCAGTCCTCACTAGCC 3853
Lbx2 GGCTGGTACGACTTGCTCAG 3854
Lbx2 GGCTCAGGTAAGGAAGGGAT 3855
Lbx2 GGCTCCTGGCTAGTGAGGAC 3856
Lbx2 GCTGCTGTCACTGAGCTGAC 3857
Lbx2 GGTCACTCACATCTCCTATT 3858
Lbx2 GAGCTGAAATAAGGCAACTC 3859
Lbx2 GGAGAGGTTGCAGTGTCTGT 3860
Ldb1 GACTCTGACCTATCATTCAA 3861
Ldb1 GGGCAAGTGGTCCCAGGACT 3862
Ldb1 GTCAAGTCCTTCTATGCCCT 3863
Ldb1 GGGACACACTCACATGGCAA 3864
Ldb1 GATTCCGATCACCTACCTGG 3865
Ldb1 GTGGTGCTGTCAGGGTAAGG 3866
Ldb1 GGAAACACACACGCACAGGC 3867
Ldb1 GGCTGTCATACAGCTCAAGA 3868
Ldb1 GGAAGAGTTCTTCCCTTCCA 3869
Ldb2 GGGAAGGGTGTTCCCTAGAA 3870
Ldb2 GTACCTGCTGTACTTCGGAT 3871
Ldb2 GAAGAGGTAAATACAAACTC 3872
Ldb2 GGAGCACAGCTCTCCCTTTG 3873
Ldb2 GATGGTTCCACATTCAGGTC 3874
Ldb2 GTGCCAGTGTTGTTGTGTTT 3875
Ldb2 GGGTGGATGTTTCTTGCAGG 3876
Ldb2 GCTAAGTCAGCGGGTTTAAG 3877
Ldb2 GTGCACAGCTGACCCAAAGG 3878
Ldb2 GCCCGGAGGAATCTTCCAGA 3879
Lef1 GGGTGCTAGGAAATGAACTA 3880
Lef1 GTCGCCAGTGCTATGCCTCT 3881
Lef1 GAACTCTAGCGAACCACTGG 3882
Lef1 GTAGAGTAAATAGAGACACG 3883
Lef1 GAGCATTTAATCTGCTGGAG 3884
Lef1 GGAGGGAGTCTGTTAGGAGG 3885
Lef1 GACTAGAAGTGAGGCGCCGG 3886
Lef1 GGGCAGAAAGTTGCCATTTA 3887
Lef1 GATTGGGCGAGTGGGATCCT 3888
Lef1 GAAGGAAAGAAGCTCTAACG 3889
Lef1 GACTTGTTCTAGGAAGTGTT 3890
Lhx1 GGTATTAATCGACTTGTTCT 3891
Lhx1 GGGTGGGAGAAAGAGTGGGT 3892
Lhx1 GGTGAAGTAACCCAGCAGCG 3893
Lhx1 GCGAGATCTGGAAGCTTGGG 3894
Lhx1 GACTTTGAAGGATGGAGGGT 3895
Lhx1 GGGTGGACTTTGGATGGACA 3896
Lhx1 GCTCGAGTCTAAGGAGAGGT 3897
Lhx1 GACCTTCCCACCTAAAGGGC 3898
Lhxl GGACTGCACCGTAGCAGCAG 3899
Lhx2 GACGCAATAGTGTCTATTGG 3900
Lhx2 GTGAAGCAGGGTATGGAAGC 3901
Lhx2 GGTGTCTGGTGGAACAGGAA 3902
Lhx2 GACTGCCCTTGGTTTCTTAG 3903
Lhx2 GTAACCGTGCCCAAGAGGCA 3904
Lhx2 GCAGGATGTGCCAGTGGCTC 3905
Lhx2 GAGTGGGAGAGCTAGTGGGA 3906
Lhx2 GACGTCGCTTTGCCCTGTCC 3907
Lhx2 GTGACTTGTCCGAAGTCCCA 3908
Lhx2 GTGCCTGACACCTACTTACC 3909
Lhx3 GTCTAACATGGAGGCTGGGA 3910
Lhx3 GTGGGTTCAGAGACAATCTG 3911
Lhx3 GAAAGGTGCACAGTCTCCAG 3912
Lhx3 GTGAGGACAAGGTAACAGCA 3913
Lhx3 GTAGTAGGAGCCCTCAGTGA 3914
Lhx3 GATGTGGAAATCCAGGTGCA 3915
Lhx3 GGTCACAGTCCTAGGGATGG 3916
Lhx3 GGTCCAGAGTGTCAGAGTTG 3917
Lhx3 GCTTCTAGGCACCTCGGTTC 3918
Lhx3 GCACCTCGGTTCCGGCTGAA 3919
Lhx4 GCTACACTGGTTTGTTTGGT 3920
Lhx4 GATGGGTCTTTACAACCAAA 3921
Lhx4 GAAACCTACCGGGTCAGCCC 3922
Lhx4 GAACTCGGAGCGCCAACCCA 3923
Lhx4 GGTGGCTGTGTGTGCTACTT 3924
Lhx4 GCAACAGTGTCTCCTCAACC 3925
Lhx4 GCCCGGGAGAGCGAGATCAA 3926
Lhx4 GTATAAATACTGCGGCGGGC 3927
Lhx4 GCCCGCCGCAGTATTTATAC 3928
Lhx4 GTCCTCTAGGATCAAGGAGG 3929
Lhx5 GCAGGTGTGTGGGTACCAGC 3930
Lhx5 GGGATTCTCCTCATGGATTA 3931
Lhx5 GGCCATCTGTCAGTGCTGTT 3932
Lhx5 GATTCTCCTCATGGATTAGG 3933
Lhx5 GTCTTGGCACAATTCCTCTA 3934
Lhx5 GACTCTGAAGGGCTGTGTGT 3935
Lhx5 GTTTGTGTGTGTGTGTGTGG 3936
Lhx5 GCGAAGCTGCCTTTGGCTCT 3937
Lhx5 GGTAAATACTTACTTAGCTT 3938
Lhx5 GIGACATCCCTGAGTCAACC 3939
Lhx6 GTGTTTGAGGAAGAAGGCTG 3940
Lhx6 GCTGTTTACATCTGTAAATG 3941
Lhx6 GGTAAATCTTGAAGTGGAAG 3942
Lhx6 GATGAGATTTACATAGTCTG 3943
Lhx6 GGAGCCTGTGCTAGTGAGAG 3944
Lhx6 GCTAGTGAGAGTGGGAGGGT 3945
Lhx6 GATACTACTTCAGATTCTTC 3946
Lhx6 GGTCAGCCCATCTACAAGGC 3947
Lhx6 GAACTCAGTCACGTAAGTGG 3948
Lhx8 GAAATTTCAGTCCAATAGGA 3949
Lhx8 GCTTCCGGGCTTAGAGAAGG 3950
Lhx8 GCTCTTTCAGCGGCTCACGG 3951
Lhx8 GATAATGAAGGGACAAACGA 3952
Lhx8 GGGTTTGGGCTGGAGATGGG 3953
Lhx8 GGAACCTCGCAGAGAGGAGG 3954
Lhx8 GCCTTTGATAGGAATCGCCA 3955
Lhx8 GAATGCTGGCTCCAGCAGGT 3956
Lhx8 GGGAAAGGAAGTGCCGGAGC 3957
Lhx8 GACACAGGCAATTATGCTGC 3958
Lhx9 GCAAGGCAAAGGCAGGCTAG 3959
Lhx9 GTGGCCTCAGAACGGGTGTC 3960
Lhx9 GGCATGGACCAAGGACTGGA 3961
Lhx9 GGGTTTCTAATGCCCAGCTA 3962
Lhx9 GAGGCTACAGTGTCTCAGCT 3963
Lhx9 GGATTATTGAGAGGCTGGCA 3964
Lhx9 GAAAGGTGGGAATGAAGCAG 3965
Lhx9 GTCTATGCGGCTCTGAGTGT 3966
Lhx9 GCAGGAAGTCTTTGGAAAGG 3967
Lhx9 GAAACTAGGTACTGGAGCAG 3968
Lmo1 GTGCTGCCCAGCAAGTCTCC 3969
Lmo1 GGCAGGGAAGTCAGGCTTTG 3970
Lmo1 GTGCAAACCTCATACATTGA 3971
Lmo1 GAGTCTAGGAGGAGAGGCAC 3972
Lmo1 GTGCCTCAGGCTTGGGAAGC 3973
Lmo1 GCTCTAGAATATCTGGGATG 3974
Lmo1 GGCATCTTAGGATTCCACCC 3975
Lmo1 GCTGCACCAGTTGGGCTGAG 3976
Lmo1 GTGGTCTCTCTTAAACTTAT 3977
Lmo1 GAGCTCTAGAATATCTGGGA 3978
Lmo1 GTAGATTTCACATACTAAGA 3979
Lmo2 GGGAGATACTTTCATGACTT 3980
Lmo2 GTTCAGCTGAGTTCACATGA 3981
Lmo2 GGTACCTTCTTCAAGCACCC 3982
Lmo2 GGGCTGTTCTTACTAAACAA 3983
Lmo2 GAGTGGTTACTTTCAGCCTG 3984
Lmo2 GGAGGACTTTGCTCAGTACG 3985
Lmo2 GTCTTTCACAACTCTTTGGA 3986
Lmo2 GCTATTGCTAGGGAGAAATC 3987
Lmo2 GAACTTGTCTTCAAGCTTGA 3988
Lmo3 GGTCCAGTTGGTTTGGGACT 3989
Lmo3 GTGTGATAGGCATGGGTGGG 3990
Lmo3 GAGCTCCAAAGGAGAAGGGT 3991
Lmo3 GAAATGCATTAAAGCTGACA 3992
Lmo3 GACCGGCTATGCCAGGACTT 3993
Lmo3 GAGCTGTTCATTTAATTCCA 3994
Lmo3 GTGGGCGAGTCCTGGAGGTA 3995
Lmo3 GCAGTAGCATAGAGTCACCA 3996
Lmo3 GATCCCTGGAGAACAATACA 3997
Lmo3 GCTTTGTTGCTAATTTCCCA 3998
Lmo4 GGTCTGGTIGGTCTTTGTGG 3999
Lmo4 GAGAAACACTAGGACTTTAT 4000
Lmo4 GAGCAGATAGCTGGGAGCCT 4001
Lmo4 GCAAATGCTCGCATCGCTTT 4002
Lmo4 GAATGTCTTGAGCAGATAGC 4003
Lmo4 GGCTTTATCTGGGATCCATT 4004
Lmo4 GCAGTTTAAAGACCTAGGGC 4005
Lmo4 GAATCTGCATTTCCTGCCCT 4006
Lmo4 GAAACTTACTTTCCCAGAAA 4007
Lmo4 GAGCTCTGCCTAGGGAAGTG 4008
Lmx1a GTTCGCTCCTGCTCTCTCCC 4009
Lmx1a GCCTCTCTAGAGGCAGGAAC 4010
Lmx1a GTCTGCCATCCAGATAGAAC 4011
Lmx1a GATGTGTTTATTGAGTCACT 4012
Lmy1a GGGAACGTCTGCAGGAGCAA 4013
Lmx1a GTTAGGAGAACGCAGTTAGG 4014
Lmx1a GAGTACCATAGTTCTAGTGG 4015
Lmx1a GGCCACATTAGTATAGGATG 4016
Lmx1a GGGTTAGGAGAACGCAGTTA 4017
Lmx1a GGGCAGCAGACTGGAGCATC 4018
Lmx1b GACAGCCTGGTGTGCTGAGA 4019
Lmx1b GGATCTGGACCGCCTTCTCT 4020
Lmx1b GGATCAGATTTGGAGCCTGA 4021
Lmx1b GGCCTGGCAGAAATAGGGCG 4022
Lmx1b GAACGCAGCGACTTCTCCAG 4023
Lmx1b GAGCCGCTCGGTTTAGAGCT 4024
Lmx1b GGCGACGGCACTATTTGACG 4025
Lmx1b GGTCCCTTAGCCACAATGAA 4026
Lmx1b GAGACCAAGAGAGTGTTAAG 4027
Lmx1b GCTCCTAGGGTCGAGGGATG 4028
Lrrc41 GGTCAACCAAAGAATTCTGA 4029
Lrrc41 GGCTCCCGACATGGGACTAG 4030
Lrrc41 GACTAGTAAGGGTCACTCGA 4031
Lrrc41 GCATTTGTCTTGTCTACTTC 4032
Lrrc41 GCTTTGTTGAGCTAGGTCCC 4033
Lrrc41 GGACCGCTCCATAAGGGATA 4034
Lrrc41 GTAAAGAGCAGAGGTTACAG 4035
Lrrc41 GGGCTCCTGGGATCAAACTC 4036
Lrrc41 GCCCAATTTGTGCGTGTGTT 4037
Lrrc41 GTTAGTCTTTAACGTAGCTT 4038
Lyl1 GGAGGAAATGCCTGGATAGC 4039
Lyl1 GGCTGGGCAAAGACAAAGTG 4040
Lyl1 GAAGGAGCCAGCTGAGGACC 4041
Lyl1 GCTCAGGAGAGCAGTTCATC 4042
Lyl1 GGCCTCAGAGGACCGGAAAG 4043
Lyl1 GCTAGAGGAGTCACTAGGGT 4044
Lyl1 GCAGTTCATCAGGTGGCCAC 4045
Lyl1 GTGGTAATGTTGTAGAAGTG 4046
Lyl1 GCTCCGGAAGGAGACAATTC 4047
Lyl1 GCTGTGCTAGAGGAGTCACT 4048
Maf GTTATTGCCACAAATCGGGT 4049
Maf GCTCTTTCAAAGGGCTGGCA 4050
Maf GGATTCTAGTGTACATTCGA 4051
Maf GTGCGAAGTTTAGTGCACCA 4052
Maf GGAGGATGGTTTGCTTTCCT 4053
Maf GATCACCTCACTTGCAGAGA 4054
Maf GTGTGCACGTTCGAGCTTTC 4055
Maf GGGTTTCCGGACTTGTCCGG 4056
Mafa GATCCCAACCGAAGATAGAA 4057
Mafa GGAGGAGGAGGGCAGGATTG 4058
Mafa GACCTCGTGCTCTAACTCAA 4059
Mafa GTCTCCTTTGGAACAGGCTG 4060
Mafa GCTGTGGTTCATCTAGGACA 4061
Mafa GGGATCTGGAATTCTGGAGG 4062
Mafa GGACACTGAGGGAAGGAGCT 4063
Mafa GGCCTGGAGTCTCCAGAATG 4064
Mafa GAGGAACAGAAGGAGGAGGA 4065
Mafa GGTGTCTCAGATCCATTAGG 4066
Mafb GGAGGCTGGACCATTGAAAT 4067
Mafb GGTTTAGATCAGTGAACTGC 4068
Mafb GGTGAGTGTGTCCTAGCTGC 4069
Mafb GGAGGAGGAAGGCAGAACAC 4070
Mafb GAGCCACTGAGTGCACAGAC 4071
Mafb GTGGCAGCCTGGAGAGAGAA 4072
Mafb GCAAACCCTCCTGGGAACAC 4073
Mafb GTGGAAACCTTACAACTCCG 4074
Mafb GTTGCGCACCGTGGCCACTT 4075
Mafb GCGGGCCGAGTGAATGTGTG 4076
Maff GTAAGGACGCGTCAGGGACG 4077
Maff GATCGGGACCGCAGTTCACT 4078
Maff GCAAGAACTCCGAGGTTTCA 4079
Maff GGTTTCACGGGTCCTGGGTC 4080
Maff GGTTTGTTTACGTCTCCCGG 4081
Maff GGTGACGTCACTGCATGACT 4082
Maff GCTCGCCTTACAACTGCGCG 4083
Maff GACAAGCACGCACTGAGCGC 4084
Maff GAAACAAGGCTACCAGACCC 4085
Maff GCTCTGAAGCCTCTTCTCCC 4086
Mafg GACCTGTGAGTTGGAGGCAA 4087
Mafg GGCTGATCCTTGCTTGCTGT 4088
Mafg GGGCTCTGGACCACTCATTC 4089
Mafg GACCGTGCTCCTGCAGAGAC 4090
Mafg GCACAGGAAAGTGCAGAGTG 4091
Mafg GGTGTATGTGTGTTGAGGGT 4092
Mafg GCCTCAGGGCTCAGGGTTAA 4093
Mafg GGAGAACGGCTCAGGAAGGG 4094
Mafg GGAGAGAAGACCTACGTAGG 4095
Mafg GCCATTCAGGGTCACAGAGA 4096
Mafk GGTGGTGGCAGTGAGGATGA 4097
Mafk GTCAGGTTAGAGGCAGAGGG 4098
Mafk GGAAGGTGCCTGGAAGAAGG 4099
Mafk GGACTGCCAGGATGTCGTGC 4100
Mafk GGTGAAGGCACTTAGGGTGA 4101
Mafk GTTTCTGGTCTCCCAGAATG 4102
Mafk GAGGCTGACAGCAGGGTGCA 4103
Mafk GTGCTGAGGAACTGCTTCCG 4104
Mafk GTAAGGAGGGAGGAGGGATT 4105
Mafk GACATCACTAATGTTGTTAT 4106
Mapk8ip1 GCTTTGTAGCCAGGATGGGT 4107
Mapk8ip1 GTGTCTATGTCCTCTCAGCA 4108
Mapk8ip1 GATCTAGCCCGTGGTGGCTA 4109
Mapk8ip1 GGATCGAAGCGTCAGCACTT 4110
Mapk8ip1 GGAGAACCACACAGCCTGGC 4111
Mapk8ip1 GAGTCCCAGACCTTACAGGC 4112
Mapk8ip1 GTCCTGCTCCATTTATGTGA 4113
Mapk8ip1 GAACCTAAAGCCAGAGGCCT 4114
Mapk8ip1 GCTCCATTTATGTGAAGGGC 4115
Mapk8ip1 GACGGAGGAGGTCACTACCA 4116
Max GGACACATCATGCCATTCCT 4117
Max GTTTCTGCACTCAATAGTCA 4118
Max GCCAGATTTCAGGGAGGGTG 4119
Max GACTTGTAGTCCTCGAGCGT 4120
Max GAGAAACTACAAATCCCATC 4121
Max GAGATGCCAGATTTCAGGGA 4122
Max GATACCAGAAGTAGAGACAA 4123
Max GAATCTAGTTTAGGCTTTGT 4124
Max GGCTGTAAGGGAGACAAAGA 4125
Maz GGAAGGCATCTCTGGGAAGC 4126
Maz GGGACAGGAGGGACTCTAGA 4127
Maz GGGTTGTTACCTCACTGAAG 4128
Maz GAAGGGAGTGGACACAGCAC 4129
Maz GGGTGGATCAAGCTCTCTGC 4130
Maz GAGGACTTGGAACAGGTGGA 4131
Maz GTTGCTGGGATCCATGGCGG 4132
Maz GAAATAACGGCCGCTGGCGG 4133
Maz GACACACAAGAGGCTGGAGC 4134
Maz GCAGCCAATCCAAACACAAG 4135
Mbd2 GCCTGTCTCAGAGATGAGTG 4136
Mbd2 GTGTACAGATGGAGAAACCA 4137
Mbd2 GAGTGGCAGAAGTGTACAGA 4138
Mbd2 GACCAGTGACCTTCATGCAG 4139
Mbd2 GTGTCGTGAAGGCAGAGGCT 4140
Mbd2 GGCTCTTGATATAAACCTCC 4141
Mbd2 GTGGCCCTGACTCCAAGGTC 4142
Mbd2 GGGAGTTTGTGCAGGAGTGG 4143
Mbd2 GCAAACAAAGGCTCTGAGCT 4144
Mecom GATTCTCAGGCAGGGCTCTA 4145
Mecom GACCAGTTCACTGAAAGATG 4146
Mecom GGCAGTTCTCTTGCCTAGTG 4147
Mecom GTAGTTTGGAAGCTCTGAAG 4148
Mecom GGCTTCCCTGCATTGATCTT 4149
Mecom GTGTTTCTGTCTTCTCTTGG 4150
Mecom GATGGCAATCGCCGAGGAGG 4151
Mecom GTGGTGGGTATTCTTAGATG 4152
Mecom GTTACTATTGGAGAGAGGCA 4153
Mecom GGGAAGTGAGAAGGGTGGAT 4154
Mef2a GATACGAGATTACCAGACAC 4155
Mef2a GAGGGTTTGTGCCCATTGCA 4156
Mef2a GATGTGCACAAAGCAGCCAT 4157
Kaf2a GCAAACAGAAGGCAGGGATG 4158
Mef2a GAAGTTACAAAGGAAGCTGG 4159
Mef2a GCTGGATCCTTGCTGTGACC 4160
Mef2a GCCCGGGAGAGAAGAAAGAG 4161
Mef2a GGTAATAAGAATGTGATGGC 4162
Mef2a GAGGACTGCAAACAGAAGGC 4163
Mef2a GCAGGGACTCAGCATTGCTC 4164
Mef2b GGGCCAGAGGAAGACCCAAG 4165
Mef2b GCAGAGGGAAAGTCACTGTG 4166
Mef2b GACAAAGCTGGAGCTGGCTG 4167
Mef2b GCTGCACTAGAATGCTGTTG 4168
Mef2b GTCCTCCCAGTTGCTCCAGT 4169
Mef2b GAATGTCAGGGTCAGAGGIC 4170
Mef2b GGAAGTAAGGCCCAGAAATG 4171
Mef2b GTCATCGCCTCTGGCTATTC 4172
Mef2b GCTCGCCTCTGGCTTTGCAG 4173
Mef2b GTGAAGGGCTTTGGGATGTG 4174
Mef2c GATACTGGGTGATGCCATTC 4175
Mef2c GTTGGCTTCAGTCTTGGTCG 4176
Mef2c GCTTGTAACTCTAAGAGACT 4177
Mef2c GCTAAACCAGGTACATTTAA 4178
Mef2c GATATCAGCAAGTGTTCAGC 4179
Mef2c GAAAGCTAGAAGACAGAGGA 4180
Mef2c GAGTTACAAGCTTTCTAATT 4181
Mef2c GTGTGATGAGAGAAAGAAAC 4182
Mef2c GAGAATGTTTCTCTACACTT 4183
Mef2c GTCATGGCACTTAAACGATT 4184
Mef2d GCTTCTGGATGTTTCCTGTG 4185
Mef2d GGAAATGACAGAGTCTGGCG 4186
Mef2d GAGAGTGAIGGACAAGCAGG 4187
Mef2d GTCCCTGTTCTGGCTTCTTG 4188
Mef2d GCCATTGGGTCCCAGCTTGT 4189
Mef2d GCAGAATAGTCCTATTGAAC 4190
Mef2d GTCACAGGTAGAGGGAGCAG 4191
Mef2d GAGGCAAGGGAGGTAGTGGT 4192
Mef2d GCCTAGCTTGCGAGATGGGA 4193
Mef2d GAACTCTCCAGATGGCGCAG 4194
Meis1 GTGTAAGACGCGACCTGTTA 4195
Meis1 GCGTCGCCGCTGAAAGAGCT 4196
Meis1 GTCAAAGCCAGAGCAAGAAG 4197
Meis1 GAGCACCGGTGAAATTCCCA 4198
Meis1 GTGAACATATGTCAACCTTC 4199
Meis1 GAGGGCTGCAAGAGAGGAGG 4200
Meis1 GCCGCATTGGTCTGGAGCTG 4201
Meis1 GCCAGAGCAAGAAGAGGAGC 4202
Meis1 GGGAATGCAAACTGCCATTC 4203
Meis1 GCAATCTAAGCCACGAGAGC 4204
Meis2 GAGTGAGTGTCAGTAGGTGT 4205
Meis2 GAACTCGGAGCATAGTCCCT 4206
Meis2 GCTCGTAACCTTCAGTTCGG 4207
Meis2 GCAGGAGCCAAGAGGAGTGG 4208
Meis2 GGGTCCTGGCCTCAATCTGG 4209
Meis2 GTCAGTAGGTGTTGGCAGGT 4210
Meis2 GCTCAAAGGGAGAGAAGGCA 4211
Meis2 GAAAGCAGCGCCTCCTGCAA 4212
Meis2 GATATAAATCCTCTCCTACA 4213
Meis2 GTTGGCAGGTTGGCTGCAGC 4214
Meis3 GTCTGAGCTAGGAAGACTTA 4215
Meis3 GAGAGGCGGTGACTTCGGGA 4216
Meis3 GGCACACTCAGGACAATAAG 4217
Meis3 GTGGTGGTGACAGAAATAAG 4218
Meis3 GACTGCACAGCCATGGCTAA 4219
Meis3 GAGCCACCTCACTCAGTCTA 4220
Meis3 GGCCTGAGAGGCTATGGAGG 4221
Meis3 GAGCTGCTGTGCTTCCCTCA 4222
Meis3 GGCTAGGCAGAGAGGACCTG 4223
Meis3 GCAGTGAGGACCAAGAGGGA 4224
Meox1 GTGAGATGGAAGGAGCCCAC 4225
Meox1 GTTCCCTGTCAAGGCCCTGT 4226
Meox1 GACATGGAGGCAGGAACCCA 4227
Meox1 GCTGACAAATGGGTTGCTGT 4228
Meox1 GAGGTGAGGTGTGCTGTCCC 4229
Meox1 GTGATTAGCCCGGAGAGGTG 4230
Mecx1 GGTAGAGAGTCTTTAAATCA 4231
Meox1 GTCTACGCTATACCTATACC 4232
Meox1 GAGACAAAGATGGATGGAGG 4233
Meox1 GCTTGTGTATGTGCTGTGTT 4234
Meox2 GTCCTGCAATTGCATGACTT 4235
Meox2 GGAACCTATGGGACAGATTG 4236
Meox2 GGGATGTCTGCAGTAGCCTA 4237
Meox2 GGTTCCAGCGTAAACACATT 4238
Meox2 GTTTGCATGTGGTCAGCGCT 4239
Meox2 GCAGCAAGGCTTTGACGGTA 4240
Meox2 GTCCTGCCAGCAATGGGAAC 4241
Meox2 GGAGCTTCCACCACAGCTAG 4242
Meox2 GATTTCATTTCTCAAAGGAT 4243
Meox2 GAGACACTGTGTGCTGGCTT 4244
Mesp2 GTATACAGCAAATTGGCTAA 4245
Mesp2 GAATGACTTCCAGCCCTCCC 4246
Mesp2 GAAGTGGAAATGGAAGGAGG 4247
Mesp2 GAGAGCCCTTGGGCAGTGAC 4248
Mesp2 GGCTGGGAAGTGGAAATGGA 4249
Mesp2 GGAAAGGCCTGGAGGTGGGA 4250
Mesp2 GAAGGGAAAGGCCTGGAGGT 4251
Mesp2 GCAATTTCAGGATTAATCCA 4252
Mesp2 GCATTGTTTCATTAGGGAGA 4253
Mesp2 GAGGCACGGGATAGACATCC 4254
Mga GAATGTCTGCCCTCACATTC 4255
Mga GGAAACCAAGAATGTAAGGA 4256
Mga GGAAAGGAGAGACAGGAGAG 4257
Mga GAAGCTTCATAAGTTCTTTC 4258
Mga GTTTGGCCTCCTGATGTTGG 4259
Mga GAGTCTTCTTGGGAAAGGCC 4260
Mga GCTCTAGAAATTGTGAGAAG 4261
Mir101a GTTGGAAAGTACCAGAACAC 4262
Mir101a GGCTTGAAACTTAACCTTCC 4263
Mir101a GTTTGAGATGTGACTGACAT 4264
Mir101a GGCAAATCACAGAATGTCCC 4265
Mir101a GCAAATCACAGAATGTCCCA 4266
Mir101a GCTATCTTTGCACTTTGGAG 4267
Mir101a GCACGTTTATGGTTCTTGAT 4268
Mir101a GTGTGAGGCTAGAAATCTTT 4269
Mir101a GTGCATAGGTGTGAGATTGG 4270
Mir101b GGAGTTCAGCAGGAGCCCAT 4271
Mir101b GGGCTCTGCAAATGGGCAGA 4272
Mir101b GAGCCCTCCCTTCCAAATTG 4273
Mir101b GTTCTGCTGCTCATGACCCT 4274
Mir101b GGAAGAGGTAAGACGCACTT 4275
Mir101b GGTGTACTGGGAAGAAGGCA 4276
Mir101b GAGCCGCTCTTGTCTTCAGC 4277
Mir101b GTCCCTTTCTAGGAGACCAT 4278
Mir101b GGTCAGATTTCCTGTTTGTA 4279
Mir101b GACCTCAATTAATCTAACAC 4280
Mir106a GGTCCAAGAGGATAGATATT 4281
Mir106a GTCTGACTCTTAAGAGTAAG 4282
Mir106a GAGAGTTAACTAAGGTGGGA 4283
Mir106a GAAGGGCAAGGCTGAGGGAG 4284
Mir124a-2 GTCTTCTTTGTGACCTGTAA 4285
Mir124a-2 GGTGCTTTAGGATGGGCGGT 4286
Mir124a-2 GATGGAAAGAAGAAGAATGA 4287
Mir124a-2 GGCACAGGTTTGGTTCACTG 4288
Mir124a-2 GTTAGATGGGTAAGGGCGCG 4289
Mir124a-2 GAGATTGGAGAATGCGGTTC 4290
Mir124a-2 GTGTTCTCGGAGGAAAGAGG 4291
Mir124a-2 GGTAGAAAGCAGAGACAGTT 4292
Mir124a-2 GACTGGAGAGGAGGGACAGG 4293
Mir124a-2 GCCAGCCTGGACCTTGACTG 4294
Mir124a-3 GCTGCCTGTGCGCTAAGAGA 4295
Mir124a-3 GGGACAGTGCCAAGGAAGCC 4296
Mir124a-3 GGGCCTTTGTTCCTGCAGAC 4297
Mir124a-3 GAAGGGTTGTCCTGGGTGTG 4298
Mir124a-3 GCGGTTCGAGAGTGTCCAAG 4299
Mir124a-3 GCTCTCTTCTCTTACGCCTC 4300
Mir124a-3 GACTGGCACCTGCAAAGGGA 4301
Mir124a-3 GGGTTGGGCATAAGCAAAGG 4302
Mir124a-3 GCTTCTGAGCCTCTCTCTCC 4303
Mir124a-3 GCACTCACGCACTCCTGGTG 4304
Mir125a GACCTCATTTCTGAGTTGGG 4305
Mir125a GACAACTGACTTTGGTCTAG 4306
Mir125a GGCCGGCAGTGTAGCTATGG 4307
Mir125a GAGACCAGAAGTAGGGAGGG 4308
Mir125a GCCTGGGATATGAAACCTTT 4309
Mir125a GAGACTGGAAGATGGGAGGA 4310
Mir125a GTCTGGGAGGTTGGGAAGGA 4311
Mir125a GCTTCCCTGGATCTGTGGGA 4312
Mir125a GCTCTGAGCCAGGTTGGTTG 4313
Mir125a GTCCAGGTTGCTCTGAGGAC 4314
Mir125b-1 GTTCAATAGGACAGAGAATG 4315
Mir125b-1 GTGTTCAATAGGACAGAGAA 4316
Mir125b-1 GTAGCTGTCTGTGAAGATGG 4317
Mir125b-1 GAGCTAAAGGTGATTAGAGG 4318
Mir125b-1 GTGTGTGGATGCCAAACAAT 4319
Mir125b-1 GAGCTGAACCTACAGAGGTG 4320
Mir125b-1 GGAAGGCTGTTGGGTGGGAG 4321
Mir125b-1 GGGTTGGAGCACGTTCAAGA 4322
Mir125b-1 GGCCATATCAGGACAAGGAG 4323
Mir133a-1 GGGACAGCTGATCTAAGTGC 4324
Mir133a-1 GTTAGTGATACATTGATGTA 4325
Mir133a-1 GAGCAACTGCACTTGCTGAC 4326
Mir133a-1 GAGTATGGAAGTCATCCTCC 4327
Mir133a-1 GCAAATTATAAAGAAGAGGG 4328
Mir133a-1 GTGAGTACATGTTAAACTCT 4329
Mir133a-1 GCTAAAGGAAACTTTCCAGG 4330
Mir133b GTTGGGTGCTTTAAAGTATG 4331
Mir133b GGTTCTCTCTGTTACAGGCT 4332
Mir133b GTACCTTGATGATTCGAGAC 4333
Mir133b GAGTCTATCGAGGGAAACAG 4334
Mir133b GAGATCAAGTGTAGGTAAGA 4335
Mir133b GAGTCCATCTGGAAGAAGCC 4336
Mir133b GACTTTAGTAGAGTCTATCG 4337
Mir133b GCATGCCACCCTATTCTTCT 3338
Mir134 GTAGGTCAGAAGTCCTCTGC 4339
Mir134 GAATGATTCGGTGGGCTGCA 4340
Mir134 GCTCTGAAAGGCTGCTAAGA 4341
Mir134 GATGGCAACTTGCAGAAAGA 4342
Mir134 GCTCTAGAAACACACTGGAG 4343
Mir134 GAGCCACAGCTGCCTCACCA 4344
Mir134 GTCTTCCTAAGAATGGATTG 4345
Mir141 GGCTCGCAGGTGGATAGTAG 4346
Mir141 GTGGAGGCCAAGTCGGCTCT 4347
Mir141 GACGCCGATGACACTGGGAC 4348
Mir141 GATCTGCCGCTTCTCTTGAG 4349
mir141 GAGATCTGCCGCTTCTCTTG 4350
Mir141 GGAGGAAGGAGCCGCTGGAA 4351
Mir141 GGAAGCCTCTGCAGGGATCA 4352
Mir141 GAAGAGTTGGCTCCCACCAT 4353
Mir141 GCGGGTCTGGTGCCAGGTAA 4354
Mir141 GGTGGGAGCCAACTCTTCCC 4355
Mir150 GGTATGGTGATACCCATCTT 4356
Mir150 GGAGTAGAGCCACTAAGCAG 4357
Mir150 GGATCCAGGTGTTCTGAGAC 4358
Mir150 GAAGACATTTCCACCGGGAG 4359
Mir150 GTGTGGAACTTTCTTTGGGT 4360
Mir150 GCAGAGGTTATGTATGGTTA 4361
Mir150 GCGGGTGAGGCTTCTCAGCA 4362
Mir150 GTTGCAGAGTCTGTGAGGGA 4363
Mir150 GACCTGTTTCAAACGAAGCC 4364
Mir150 GGCATATCACCATTTCTCTG 4365
Mir150 GCTTGGAAATTTCCAAACCA 4366
Mir155 GCCATATTATTGACCCATTA 4367
Mir155 GCCACATAGTGAATGGGACC 4368
Mir155 GCAGGTGCTGCAAACCAGGA 4369
Mir155 GTGATATGCCACATAGTGAA 4370
Mir155 GTTGCATATATTCTCCCTAA 4371
Mir15a GTTATCCTAAGATGATGTTC 4372
Mir15a GTGGTTTATATTCTGGCCTA 4373
Mir15a GAACATCATCTTAGGATAAC 4374
Mir15a GAAGCTTTGTCCIATGGATT 4375
Mir15a GACACTCAAAGGACAGTGTC 4376
Mir15a GCTGGCACACTTGAAAGCAA 4377
Mir15a GGAAACAAATAGAGTTGAAG 4378
Mir15a GCGTGCTGGAGGAAGTGCTT 4379
Mir16-2 GCATATGTGTGTAAAGAGTC 4380
Mir16-2 GTTAAGGGAGAGGCAAAGAG 4381
Mir16-2 GAGGTCTTGTTCGCCTTCCT 4382
Mir16-2 GGCTGAAATTTGTGTTTGCT 4383
Mir16-2 GAGGCTCTAGGTTAAGGGAG 4384
Mir16-2 GCTGGATAACAGAAGTTTAG 4385
Mir16-2 GCTCCTCACCTGGAGGCTCT 4386
Mir16-2 GCTATCTCTGTAGGCGGTTC 4387
Mir181a-1 GCATTGATCTGACAAATGAG 4388
Mir181a-1 GATTCCAGAATGACTGGAGT 4389
Mir181a-1 GCAAAGCACCGCAATGTGAG 4390
Mir181a-1 GATTACAGGACAAGTGTCTC 4391
Mir181a-1 GAATTTCAGGCAGTAGGCAT 4392
Mir181a-1 GTTACAGGCTGTTAAAGACA 4393
Mir181a-1 GTAAGAGAATAACTTCAGGA 4394
Mir181a-1 GATCTGACAAATGAGAGGGA 4395
Mir181b-1 GGTCCTTAGAATATGAGAGC 4396
Mir181b-2 GCAACCAAGCCAGCCTTAAG 4397
Mir181b-2 GAATCCCAAGGTACAGTCAA 4398
Mir181b-2 GAACTCTGGTGTTCAAGTTC 4399
Mir181b-2 GAGCATCACTAGCACTTCTG 4400
Mir181b-2 GTGTCATTCTAGTCAGAAAT 4401
Mir181b-2 GTGCTAATTTAAGGAATTCT 4402
Mir181b-2 GCAACATATCCAACCAATAC 4403
Mir192 GAGTTGCTGTTACAGAGGGT 4404
Mir192 GGAGTTGCTGTTACAGAGGG 4405
Mir194-2 GAAGGCTTGGCTTAGGGCTC 4406
Mir194-2 GGAAGCCTCTAGAGTATGCT 4407
Mir194-2 GCCAACTGGCCGAGAGAGTG 4408
Mir194-2 GATCAAGGCTTAGACAGAGT 4409
Mir194-2 GGCAGCTCTGCTGCTTCTCT 4410
Mir194-2 GGGAGCCTTCAGCAGCCTTC 4411
Mir194-2 GATGGCTTGGCAGGAAGGCT 4412
Mir194-2 GGGTCCAGGAAGTACCAGAC 4413
Mir194-2 GGGATAGATGCCATGTGGGT 4414
Mir194-2 GAGAAGCAGCAGAGCTGCCA 4415
Mir196a-2 GAGAGCAAACTGCAATCTTG 4416
Mir196a-2 GATAGTCTCCCGTTAGTTTC 4417
Mir196a-2 GAGGGTTTAGTCTAGACACT 4418
Mir196a-2 GGAATAAACTTAACTGCCGG 4419
Mir196a-2 GGCTGACAGCAAAGAGCGGA 4420
Mir196a-2 GGGAAAGACAGAGAGAGGGA 4421
Mir196a-2 GTCAAATGCACCCGATTAGA 4422
Mir196a-2 GGAGCAGGACAACTTGGAGG 4423
Mir196a-2 GCGGCAGCAAGAGAAGGAGG 4424
Mir196a-2 GAACCGAGAGAATCGGATCC 4425
Mir196b GGGCTGGGTTTGCTGCCTCT 4426
Mir196b GCGTGGGTTCTTCTGGGACC 4427
Mir196b GGCGCCTAGGAGGGAGAAGA 4428
Mir196b GGTGTCTGGCCTGAGGTCAA 4429
Mir196b GAACCCACGCCCGAAATCCG 4430
Mir196b GGAAACTCAAAGGTGAATGA 4431
Mir196b GTATGGAAGCATGGACATTC 4432
Mir196b GAGGACCGGGTGTGGATTTG 4433
Mir199a-2 GCAGGTACAAATAAGTTGTT 4434
Mir199a-2 GGCTTCCTACAATAGCGTGG 4435
Mir199a-2 GGCACATTTGCAGCAGACTA 4436
Mir199a-2 GGCCTCCTTCTCCTTCTTTA 4437
Mir199a-2 GGGTGACATCATCCCATATA 4438
Mir199a-2 GATTCTAGCGGTCTCTCCAG 4439
Mir199a-2 GGGCTGGAGAGTCCATATAT 4440
Mir199a-2 GGACTAGGCATAGAAAGGGA 4441
Mir199a-2 GGACTATTTGAGAGTGGTTA 4442
Mir199a-2 GGGAATGATGACCAAGAGGA 4443
Mir1a-1 GCTCCCATTGCGTCCGCACT 4444
Mir1a-1 GTGTCTCCAGCTCTTTCTGT 4445
Mir1a-1 GTAAAGACTGGAAGCAGACA 4446
Mir1a-1 GTAAGTTTAGCCACAATCTC 4447
Mir1a-1 GGCACTGAGACCTTCTCTCG 4448
Mir1a-1 GGACTGATGGATCAGGAACT 4449
Mir1a-1 GGATGTGACTTCCCTCTGTT 4450
Mir1a-1 GTCGTAAGGAACCGCTCCCA 4451
Mir1a-1 GACACCCACTGCAGGAGAGG 4452
Mir1a-1 GAGTTCTCAGGGAGCCTAAG 4453
Mir200a GAGGAAGGACTTAGCACCCA 4454
Mir200a GACGGACTTGGGATGAGGAG 4455
Mir200a GCATCTACTAGGCTTAGTTT 4456
Mir200a GATCAAGGCACTCTGGAAAG 4457
Mir200a GTCCCAAGTATCCTTGGGAC 4458
Mir200a GGTCTGCTTTGTCCAAAGCA 4459
Mir200a GCGGCTCCATTGCTGCATGC 4460
Mir200a GCGGCCTCCATATCCAACTT 4461
Mir200a GGATACTGGGATGAGGGACC 4462
Mir200a GATCCGAGGAAATCAGTACA 4463
Mir200b GTTGGAACTGCGTGTCTTCA 4464
Mir200b GTCATCTTCAACTCCCTGCT 4465
Mir200b GCCTGCCTCCCAGCTCTTTC 4466
Mir206 GCGTCACTAACTGTGAGGCC 4467
Mir206 GTCTGACTGATCACCCTGGA 4468
Mir206 GGCAGCTGTTGAGCCATTCA 4469
Mir206 GATCTCAGACTGAAGTGTAT 4470
Mir206 GCCTAACAGGCAGAGCTTGT 4471
Mir206 GACTAGTATGCTAGTATGCC 4472
Mir206 GAACAGCCTTGGATCAGTCC 4473
Mir206 GGCCAAACTTCCTGCACATT 4474
Mir206 GACCAATCCACCAAATGTGC 4475
Mir21 GCAGAGACGGACCTATGCCG 4476
Mir21 GTTAGAGCCCTCCCAGTGTA 4477
Mir21 GTTTCCTCGGTTCAACACTA 4478
Mir21 GAGATCTAAGCGGGACTATG 4479
Mir21 GGCCCTGTGAAGGTATCAGA 4480
Mir21 GGGACAGTCAGAGAGAGGGA 4481
Mir21 GAGCCCTCCCAGTGTAAGGC 4482
Mir21 GTTCTGCTTTCTTTCCTACA 4483
Mir21 GCAGGAGGGATCCTCACCTG 4484
Mir21 GCCTGAGAGAGCTACCTCCA 4485
Mir218-2 GACTAAGAGAAGGAAGGAAA 4486
Mir218-2 GGTCCTGTAAACACCAAGGC 4487
Mir218-2 GTACTAATCACGCTCAGTGG 4488
Mir218-2 GGATCCTTTGGGTACAACAC 4489
Mir218-2 GTGAGGGCCTTGGTATGAGT 4490
Mir218-2 GGACACAACCTCTGATGGGA 4491
Mir218-2 GAAGCCAGACGCCCTACCCA 4492
Mir218-2 GGAGAAGCTGAAGCCAGAGC 4493
Mir218-2 GCTAGGTCACTGCCATGGTG 4494
Mir23b GACATTATCGCTTGCCATGG 4495
Mir23b GGGCTAGAGCCACTTTGAAT 4496
Mir23b GTCTGCAGGAGGCAGTGAAG 4497
Mir23b GGTTCTCTGACCTGTAGAGT 4498
Mir23b GACAATGGAGACAGAGTAGA 4499
Mir23b GAGGGCTGCCAAACGGTCTT 4500
Mir23b GCAGGTGTGGTGTGTAGGGA 4501
Mir23b GACAGAGTCAAAGTGAGGGC 4502
Mir23b GGAGAACAGGGTGTGTCCCA 4503
Mir6a-2 GGCCTAAGGAACACTTGTGC 4504
Mir6a-2 GATGTCTGCATCACTGTCTC 4505
Mir6a-2 GGTCTCTCACCAATGCCTCG 4506
Mir6a-2 GATTGGGCTTACTTCTTGTT 4507
Mir6a-2 GGCAGTTTCCCTTTGAGGCA 4508
Mir6a-2 GTGTTGGCTAGAGGGAAGTG 4509
Mir6a-2 GATGTGGGCTAGGAGGGACT 4510
Mir6a-2 GATCGGACTGTGTGAGACAA 4511
Mir6a-2 GCTGGCTAAGAACTGCTCAG 4512
Mir6a-2 GGGTATCTGTGACTCCAGGG 4513
Mir375 GCCATTGGGAGGTGAGCAGC 4514
Mir375 GGATGCACAAGAAGCTATGT 4515
Mir375 GTTCTTAGTTTGGCCAGTGG 4516
Mir375 GGGCAAATATTGACTCATGG 4517
Mir375 GCTGACACCAGCAAACAGTC 4518
Mir375 GATGTTCTGCCTTCGCTAGG 4519
Mir375 GAGTGCTCTGAGTCCTGGCT 4520
Mir375 GTCAGCATGCACAGGTCAGG 4521
Mir375 GGTGGTAGGGCAATGATGCG 4522
Mir375 GTGGGAAGATTCTATCTCCA 4523
Mir7b GAAGGCCAACTGGACTGTTT 4524
Mir7b GGACTCTGAGTCCTTGAACT 4525
Mir7b GGAGGGTAAGTCAGTGAGTG 4526
Mir7b GTGAGAGAGACTGTGTTAGA 4527
Mir7b GCACTTGAGGGTGTTGAACC 4528
Mir7b GGTGTTGAACCTGGCGGAGG 4529
Mir7b GGTTCATTCTATACACCCTA 4530
Mir7b GAGGGACTCGGAGCAGAGTT 4531
Mir92-2 GGAGGGAAACCAAGGTAGGT 4532
Mir92-2 GGCCTCTGATTAAATCACCA 4533
Mir92-2 GTAATGTGTCTCTTGTGTTA 4534
Mir92-2 GAGCGGGTCCTGTGTGTCAC 4535
Mir92-2 GTGGTGCTGCGCGGACACTT 4536
Mir92-2 GCTCTCCTAGCTGGTGGAGG 4537
Mir92-2 GCACTGTTAGCACTTTGACA 4538
Mir92-2 GATGGAATGTTTGTGTTGAT 4539
Mir92-2 GAGCTTTCTCTGGAGGGCTG 4540
Mir92-2 GTTGTGTAGAAGAACAAGCT 4541
Mirlet7a-2 GGAACATACCATGGTACGGC 4542
Mirlet7a-2 GACCCATACAACTCTGCAAG 4543
Mirlet7a-2 GAAGACTGTGCAAGAGACTA 4544
Mirlet7a-2 GAGGCCAGGTTGAAAGATTG 4545
Mirlet7a-2 GGTTTGAGATTGCTCCGTGG 4546
Mirlet7a-2 GTTGTATTGTAGATAACTGC 4547
Mirlet7a-2 GTTTGAGATTGCTCCGTGGT 4548
Mirlet7a-2 GGTCAAAGATTCAAAGAAGC 4549
Mirlet7b GGAATAGCTAGAGACCACAT 4550
Mirlet7b GTCTGAGGCCTGAAAGAAGC 4551
Mirlet7b GCCCAGGTGAGAAGGCTGAG 4552
Mirlet7b GGTAAAGACATCTAAGCTGA 4553
Mirlet7b GCTAGTCGTTAGGGACAGAC 4554
Mirlet7b GCTGCCTGGCTTCCTAGGTC 4555
Mirlet7b GGCCCAGGTGAGAAGGCTGA 4556
Mirlet7b GCCTAGAGAAAGGCCAGATG 4557
Mirlet7b GCAGCAAGGCAGAAGAGGCG 4558
Mirlet7b GAGGCGTGACAGTAGACGCT 4559
Mirlet7i GGTGTTGCACTGCCTTATCT 4560
Mirlet7i GGCGCTGTAAAGATGGCGGC 4561
Mirlet7i GCAAGGATGCAGAGAGGAGA 4562
Mirlet7i GTATGTATGAAACGTGTAGG 4563
Mirlet7i GGACTGGGTGGGTGTGAGGT 4564
Mirlet7i GGCAGTGCAACACCGGAACC 4565
Mirlet7i GAGAGTAGGGAAACCAGCCG 4566
Mirlet7i GGGCGCTGTAAAGATGGCGG 4567
Mitf GAAGTCAGCAAATGGTGGTG 4568
Mitf GACACTCCTGAAAGTTGGGC 4569
Mitf GACACACTGGAAGTGGAATC 4570
Mitf GCCATAAGCAGTCAGAATAT 4571
Mitf GTGGGATGGACAGATGGAAA 4572
Mitf GGGCTGTGTTGGGAAGAAGA 4573
Mitf GAATTGTTACAGGGAGAACC 4574
Mitf GTCTGGTCTGGACACCTCTT 4575
Mitf GTAAGCTGTCTGTTGAGACT 4576
Mitf GCTGACCTCAGCCTGGTAAA 4577
Mixl1 GCGCCTTTGATGGTGACAGG 4578
Mixl1 GGGAGGCGCGAACTTGAGTC 4579
Mixl1 GAATTCTTCAACCTGCTACG 4580
Mixl1 GTAAGGTCTAGCACATAGCA 4581
Mixl1 GCTTGACCTGTCCACCAGCT 4582
Mixl1 GCTAGGCTGTTTAACCAACC 4583
Mixl1 GAAGAAGAAAGAAAGGGAGA 4584
Mixl1 GGGCAGACAGAAGGTGGCAG 4585
Mixl1 GGATTGGTGGTTGGACTGGC 4586
Mkl1 GAACCACGAGTGTACGCTAT 4587
Mkl1 GGGAAGGATGAGACTGCCCT 4588
Mkl1 GGCAAATAGCAGTTGGATTC 4589
Mkl1 GACCTCCTCCCACCTCTTGG 4590
Mkl1 GTTAGGGCTAGCCCGATTTA 4591
Mkl1 GCTCTTAAACACCGTGTTCT 4592
Mkl1 GGCAGAGAGAGAGGCGTCAT 4593
Mkl1 GTGCTTCACCAGAAAGAGTC 4594
Mkl1 GGCATTTATTGTGTCCTTTC 4595
Mkl1 GAAGTCTGGAACTGGCGGAG 4596
Mlx GCGGCTTAACTGTCCCACTT 4597
Mlx GCAATGAGGACACAGCTAAT 4598
Mlx GATGACACACGGGTCAGGAA 4599
Mlx GTTCAGGAACTTGTCTGTGG 4600
Mlx GCATCTGACTGAGTTCCTGG 4601
Mlx GGCCCATAGGGATCCAGCAG 4602
Mlx GACTGAGCCTCGCCTCTTCC 4603
Mlx GAACAGGTACTAGCCAGAGA 4604
Mlx GGTCCAGATACCTCAGTCTC 4605
Mlx GAGGCTGAAGCAGGTTTCCC 4606
Mlxip GGACTCAGTTCCGGGTATGG 4607
Mlxip GCACTCCACGTGGTGGGTAG 4608
Mlxip GCTGAAGTTGTTGGGTCTGG 4609
Mlxip GTTTAAGAGCGGTGATGCCC 4610
Mlxip GTGGCTGAAGTTGTTGGGTC 4611
Mlxip GGCACTCCACGTGGTGGGTA 4612
Mlxip GGAGCTTGGGAATAGCCCTG 4613
Mlxip GGAGAAAGCTGGCCTAATGT 4614
Mlxip GAATTGCAGTAAAGACAACT 4615
Mlxip GCCCAGAAGCCAAATTCCAA 4616
Mlxipl GTTAGACTGTAGAGAGGCAC 4617
Mlxipl GGCTGTGAACTCTGGGCATC 4618
Mlxipl GGACAATCATAAGAGCGCCT 4619
Mlxipl GGCCTCTCTTTCCCACTAGA 4620
Mlxipl GGAGAGCAACCGATGGTTGG 4621
Mlxipl GGCATCGGGTACTAGAGGGC 4622
Mlxipl GCTAACCTTTCCACTGGGAC 4623
Mlxipl GAACTTTGCTGTAGAGGCAT 4624
Mlxipl GACATAGCTAACCTTTCCAC 4625
Mnt GGAAATGGAGACATGCCAGT 4526
Mnt GAGGAATAGCACAAGACAGA 4527
Mnt GCCTGGTGATCTAGCCTAAT 4628
Mnt GGGAATTGCGACAGACCGGA 4629
Mnt GTCTGGGTCAGGAGGGCAAC 4630
Mnt GTATGTTTATAGGTAAGACC 4531
Mnt GCACTGGAGCTGTAAGTGTG 4632
Mnt GAAGAGGGAAATGAATGGGA 4633
Mnt GGGAGGGTAATGTAAAGCAG 4634
Mnt GGAAGGGTGAGACACCTACA 4635
Msc GGCTTTGTTAACAAACAGAC 4636
Msc GAGTAATGAACTTGAATGAC 4637
Msc GATTGCTTAAACTTGACTGT 4638
Msc GGTGCAGGCAGAAAGATGGA 4639
Msc GTAGTGAGCAGCTGCAGCTT 4640
Msc GAAGTATCATAGCAGGTGGC 4641
Msc GATGTGTGTTTGCTTATCCA 4642
Msc GGCAGAAAGATGGAAGGCAG 4643
Msc GGGCTGCTTGGTAGTCCTTT 4644
Msx1 GTTATTTGTCAGAGTAGCAA 4645
Msx1 GCCGATTTACACTCTGCGCT 4646
Msx1 GGGTAATTATCCGAGCACGG 4647
Msx1 GGAGGTATATCTTTGGTGCA 4648
Msx1 GCAACTGTGTAGACAACTTC 4649
Msx1 GATGCCCACCTGACTTAGCT 4650
Msx1 GAGCCTCACATCTGCCCACA 4651
Msx1 GGGCTGCCGTGGCCATTTAG 4652
Msx1 GAGGTGATTGGCGGCTCACC 4653
Msx1 GAGCAAAGAGGCCTAGCCTC 4654
Msx2 GAGAAGGCTGTAGACGGGCC 4655
Msx2 GCCAGAGCTTGGTACTCTGG 4656
Msx2 GCACCAGAAACACTTTAAAG 4657
Msx2 GAATGTTGGAAATCTGCGGA 4658
Msx2 GCCTAGAGAGGAGACTCAAG 4659
Msx2 GACTGTATCTCTGCCTAACC 4660
Msx2 GGTGCTGGAGGGAGTATTTA 4661
Msx2 GACACTGAAAGGGAAACGGT 4662
Msx2 GTTGGAGAGGCGCCTGGCAA 4663
Msx2 GAGGCGCCTGGCAAAGGGAT 4664
Msx3 GATGAGTGTTTACCAAGGAG 4665
Msx3 GGGCTAGAAAGACGCGTCCT 4666
Msx3 GTCGACAGCAATGACTCATT 4667
Msx3 GATGCAGTCTTTCCTTGACC 4668
Msx3 GTCATCAGTTTGTGGACAAT 4669
Msx3 GTCCATTCCTCCACTCCAGA 4670
Msx3 GCCTCAGCCTTCTGGAGTGG 4671
Msx3 GATCTCTTGAGGTCGAGTTG 4672
Msx3 GGGCTACAGGGTAGGAGTGG 4673
Msx3 GGGCTGAGTCTTCAATGGTG 4674
Mtf1 GCTCAGGTAGAAGAAACAGG 4675
Mtf1 GCCACAAAGGACTAGCTGCC 4676
Mtf1 GAACTGGIGAATAAACTCTT 4677
Mtf1 GCCTTGGAGTTGAGCAGAAA 4678
Mtf1 GATGCTCAGTACGGGATATG 4679
Mtf1 GCTTGGACAGTGGAAGCATC 4680
Mtf1 GTTCTTGAGCTGAAACAGGT 4681
Mtf1 GCAAGGGAGAAGAGAAGGGA 4682
Mtf1 GGTTCCAACCTTCCTTAAGG 4683
Mtf1 GGACTAACTGAAGTCCCTGA 4684
Mxd1 GGCAATGACCTCCACCCAGC 4685
Mxd1 GTTATAGGAGAGGACTGAGC 4686
Mxd1 GTGACGTCATCGTAGCCGGG 4687
Mxd1 GACAGTGGGCAACAGGTCGG 4688
Mxd1 GGGATGGAAGGGATGGCCTC 4689
Mxd1 GCGGTTTGAATTTAGTTCTG 4690
Mxd1 GAGGTGACGTCATCGTAGCC 4691
Mxd1 GAGTATCTAGCGCCATCTAC 4692
Mxd3 GGTAGCCATACCTATGAGTC 4693
Mxd3 GAGCAATCTGTAGCAGGAGA 4694
Mxd3 GGCTGTTACCTATACCTCCT 4695
Mxd3 GCTCCTCCTTCTCTTCCAAG 4696
Mxd3 GCATCAGTTCTACTGCAGCA 4697
Mxd3 GTAACAGTTTGTAGACTGAA 4698
Mxd3 GAAGGACGGGAGAGCTAGGA 4699
Mxd3 GTCCTGTCTGCCTCTGCTAC 4700
Mxd3 GGCCCTATCTACATATTCAT 4701
Mxd4 GCGTTCGGCCAGTCCCTATT 4702
Mxd4 GCAGAATGAGCTGGCTTCCC 4703
Mxd4 GACAGCAAGCCTGCTGCTCA 4704
Mxd4 GATTGTGGGCTTGGTCAGAG 4705
Mxd4 GTAGGCATTGCACGCCGATT 4706
Mxd4 GTGCCATCTTCCCTCACAAA 4707
Mxd4 GATCCGTGAGTGTCTGTTTG 4708
Mxd4 GACATGTGTGGCTCACACCC 4709
Mxd4 GTCTGGGCTCTGTCTATACA 4710
Mxd4 GTTTGCGGTGCTTGGTCTGA 4711
Mxi1 GGTGTGTCCACGCATACATG 4712
Mxi1 GGGCGGGACTACATTTCCCA 4713
Mxi1 GCTAGGATTTGCGGAGAGGC 4714
Mxi1 GTCTCCAGGCTACCCTGTCC 4715
Mxi1 GGAGGGAAGAAGAGGTTCCT 4716
Mxi1 GGAAAGACTACATCTCCCGG 4717
Mxi1 GACTTTATTTACTGAGAGGG 4718
Mxi1 GTCTCTGGGCTGGTGAGGAC 4719
Mxi1 GATCATCCGCACCCGCTCCA 4720
Mxi1 GAATGAACCTACAGGACGGA 4721
Myb GGATTCAAGAGGCTCAGGAA 4722
Myb GTCCAGCAAGTGTTTGACGC 4723
Myb GTGAGTGTCCCAAGTGCTTT 4724
Myb GGAAGAGAATGCTTCTGTAA 4725
Myb GGATGCAATAGATGCAACTT 4726
Myb GGCGTGTGTCTAAGTGAGGG 4727
Myb GTGGTAGGCACCTCCTAGGG 4728
Myb GCTCCCGGGTGTGTTGAAGT 4729
Myb GTTCAAGACTTGTGCTGACT 4730
Mybl1 GCCGTTTGAATCTGCGCACG 4731
Mybl1 GGCCAGTTTCCTTGTCCTTT 4732
Mybl1 GCTGTGAGTCTCGCCACTTA 4733
Mybl1 GGTGACAGGACACGGAACGC 4734
Mybl1 GAGTAACTGAAATCTTGCAT 4735
Mybl1 GCAACTTCTCAACAGTTACA 4736
Mybl1 GAAGAACACTTGAAGTTCTC 4737
Myc GACGAACGAATGAGTTATCT 4738
Myc GGATACCGCGGATCCCAAGT 4739
Myc GAGCTCCTCGAGCTGTTTGA 4740
Myc GAATTGCCAACCCAGATCTG 4741
Myc GATGACCGGAAGCTTGTCTT 4742
Myc GAAGTCCGAACCGGAGGTGC 4743
Myc GCCCGAACAACCGTACAGAA 4744
Myc GACGAGCGTCACTGATAGTA 4745
Myc GCCTTGGCTTCAGAGGCTGA 4746
Mycl GACTGTTCGAGAGGCTCCCG 4747
Mycl GTCTAACTACTCAGAACTAC 4748
Mycl GCTAATGGTTACTGAAGCAA 4749
Mycl GTGCGTCCCACCCATGACAG 4750
Mycl GAACACTATCAAGATCTCGC 4751
Mycl GGGAAGTAGACTAGCAGGGT 4752
Mycl GGACGCACCTGAACCTGGTG 4753
Mycl GACCAGTTCAGCCAGGAGGT 4754
Mycl GAGGATGAATTCTGGGAGGC 4755
Mycs GACCTGGTGGGTGGATTCAA 4756
Mycs GCTCTCAAGAGCATCTTCCC 4757
Mycs GGCACAGGACATGATGCTCC 4758
Mycs GCACAGGACATGATGCTCCT 4759
Mycs GTGGATTCAAAGGAGGGTGG 4760
Myef2 GGTTAAGGGAATGATCACTT 4761
Myef2 GCTAGTAATAGTAACCAGAT 4762
Myef2 GGAGAATTTAATTCCCTCAC 4763
Myef2 GCCTTTGGATGAGAGGACTA 4764
Myef2 GCTTATGATAATCTAGAACT 4765
Myef2 GTGAATTCATAAAGAGCTAA 4766
Myef2 GGACACTAGAGCTCTGCTGG 4767
Myef2 GAGTTCAATGTTGCCTTCTG 4768
Myef2 GAACTGAAATGCCTCAGCCG 4769
Myef2 GGGACAATTTAGCTGGAAGA 4770
Myf5 GAACAATAAATCAACCGTGC 4771
Myf5 GGAAGGATGGAAGCTCGGAG 4772
Myf5 GGGAAGGATGGAAGCTCGGA 4773
Myf5 GGAGGTTGGTCCCTGTAGCT 4774
Myf5 GTCCCAAAGGGCCCTCCACA 4775
Myf5 GACACGTGTGCTGGGAAGGA 4776
Myf5 GTTTGTGTACTGGTAACAGT 4777
Myf5 GGTTAGGGCTGTCTTTGGTA 4778
Myf6 GAATCCTAAGCAACCAACTT 4779
Myf6 GAATTCAGTTGAACTCTGGA 4780
My46 GGGCTGGAATTGGAGTGTGT 4781
Myf6 GTGAACTAATGTTTACTGCA 4782
Myf6 GGTATGCAACCGCATTAACT 4783
Myf6 GAATTATGAGAAGACAGAGC 4784
Myf6 GATGACTCTCTGTCTTGATA 4785
Myf6 GCACTAATTAAATGCCATCT 4786
Myf6 GTGTTAACTATAAGCTGTTT 4787
Myocd GGTACATCTCCAGAACGCGC 4788
Myocd GATGGATGGGTAGGGAGGCA 4789
Myocd GCTTACTGCAGGGCTCTGGA 4790
Myocd GCAGCTGACTTCTGCCCTCC 4791
Myocd GGAGTGTATCTGCTTGTCCT 4792
Myocd GTCTTTCTGACCCAGAGGGA 4793
Myocd GGTCCCTTTCCCACTATGAA 4794
Myocd GACTAATCTCTGCCCTGATC 4795
Myocd GTTTCACAGAGTTTCCTCCA 4796
Myocd GGCAGCCTATGACATCAGCC 4797
Myod1 GCTGGTTATGCTATGCAAGC 4798
Myod1 GCAAAGCCAGAGAAGGTTGC 4799
Myod1 GCATGAACATCCCAGGGTTG 4800
Myod1 GAGCTGGAAAGGGAGGCTGG 4801
Myod1 GGTGCTCATGGCCACTCAGA 4802
Myod1 GGAGCCATTAAGAAGAATGG 4803
Myod1 GGACAGAAAGGTGATCCATT 4804
Myod1 GGTCTCCAGAGTGGAGTCCG 4805
Myod1 GGATGTGGAAATGTCAGTGG 4806
Myod1 GAGATCTGGCAGAGGGCTCT 4807
Myog GCTGGGTGAAAGGTGGCCAG 4808
Myog GCTGGTGGACAGGGCAGGAA 4809
Myog GCGTTGGCTATATTTATCTC 4810
Myog GTCCAAGGCAGCTGGTGGAC 4811
Myog GCAGGAAGGGAACAAGAAAG 4812
Myog GAAAGGAGCAGATGAGACGG 4813
Myog GATTGAAGTAAGAGAACACA 4814
Myog GCTTCTTCACTTTGAGGAGG 4815
Myog GGCAAAGACAGAAACCCAGA 4816
Myog GAGAGAGTAGGCAGGAGGCC 4817
Mzf1 GTTGTATCTGACCTGAATTC 4818
Mzf1 GCAAACCAGGAAGTCTCTTA 4819
Mzf1 GTGAGACATCGAAACTCTAG 4820
Mzf1 GATTAGAACCACAACTCTCA 4821
Mzf1 GCTTTCTGGGAGTCGTAGTT 4822
Mzf1 GAGGTAATGTTTAAGTAGTC 4823
Mzf1 GCGGGACTCCATGGTAACTA 4824
Mzf1 GTTTGGTCCCTTAGTTACCA 4825
Mzf1 GGAAGAAGAGAGAAGCAGAA 4826
Mzf1 GGTAACTAAGGGACCAAACC 4827
Nab1 GTAAGGTAACAATTATGGAG 4828
Nab1 GTGTCCTCAGAACTTAACTT 4829
Nab1 GTCCTTCCTTGTTTATGTTC 4830
Nab1 GGAGTTGCTGTTGAAGTCAC 4831
Nab1 GGAGCATAAACACTGACAAT 4832
Nab1 GAGTTTAGGAATGGGAAGGA 4833
Nab1 GCCCAGAACATAAACAAGGA 4834
Nab1 GGTATCCTTAAGGCTCTTTC 4335
Nab1 GTGGAAAGGTAGAGGTTAAT 4836
Nab1 GGCCAGCCAGGAAGTGGGAA 4537
Nacc1 GTTGGATCCTGTGAGCGGAA 4838
Nacc1 GAGTCAAGAACAGAAGAGTG 4839
Nacc1 GTTTGTCCGGGTGTGTGTGT 4840
Nacc1 GGAACAGTTTAGGCTCTTTG 4841
Nacc1 GCCTGAACCTCCACTCACTC 4842
Nacc1 GATCACAGCACACCTGGAGG 4843
Nacc1 GTCTGTGTGAATACAACTAC 4844
Nacc1 GCAGTAAGGAAGGGACTTTA 4845
Nacc1 GAGCCACTCAGACTGAGTGT 4846
Nacc1 GAACCGAAGCGCTCGAAGCG 4847
Nanog GTGGGAAGTTTCAGGTCAAG 4848
Nanog GGGAAGTTTCAGGTCAAGTG 4849
Nanog GCTTTCCCTCCCTCCCAGTC 4850
Nanog GTGAATTCACAGGGCTGGTG 4851
Nanog GCGCTCTGCGTTTCTCCAGC 4852
Nanog GGAAGTTTCAGGTCAAGTGG 4853
Nanog GGGATTAACTGTGAATTCAC 4854
Nanog GCTGTAAGGTGACCCAGACT 4855
Nanog GGAGGGAGGGAAAGCTTAGG 4856
Ncoa1 GGGACGCTAAGGGACACTCT 4857
Ncoa1 GTACCACTCACTGTTCTCTC 4858
Ncoa1 GTGGTACTGTAAAGAAGGTG 4859
Ncoa1 GAGAACAGGTAGAAAGAATG 4860
Ncoa1 GACCAGGAAACAGACTCCAC 4861
Ncoa1 GTCTTAAGGAAGTGTGAGAA 4862
Ncoa1 GGAATGAACACAGGGATGGA 4863
Ncoa1 GCTCATTTGTAAGCACCAGA 4864
Ncoa1 GTCCCTTAGCGTCCCTGAGC 4865
Ncoa2 GTCCTCAGCATCTCCCTGGC 4866
Ncoa2 GAAGAAATCTAAGTGGCAAT 4867
Ncoa2 GAGCGGTGACAGCGTTCGCT 4868
Ncoa2 GCTGTAACAAATGTTAACAT 4869
Ncoa2 GGTCTAGGGACCGTGACCTA 4870
Ncoa2 GGGATTGCCTGACAAAGCAA 4871
Ncoa2 GACAGGAGAAGAAATCTAAG 4872
Ncoa2 GCTTAGTCTGGAGAATGAGA 4873
Ncoa2 GTGCACTGAGTAACACAGCA 4874
Ncoa3 GCAGGGATTTAAAGCCAAGT 4875
Ncoa3 GAGGTTCTGCTGTCACCTCA 4876
Ncoa3 GCCTGTGACTTGTGTTTCCT 4877
Ncoa3 GATGGTGGCAAGGGCATGTG 4878
Ncoa3 GGCAGACATGCCGCTGCTTT 4879
Ncoa3 GAGTGAGGTCTCAGAACAGA 4880
Ncoa3 CATGTAAAGAACAGACCACC 4881
Ncoa3 GTACAAGAAGGCTGTGTGCA 4882
Ncoa6 GCTCTTACATGAAGCTACTT 4883
Ncoa6 GGAAACTACCTATAGATATT 4884
Ncoa6 GGCTCTTACATGAAGCTACT 4885
Ncoa6 GCTTTCCTTTCAGTGCAGGT 4886
Ncor1 GTTTGTTCTTTCTCAGATGG 4887
Ncor1 GCATGCTTGCTTACTGTGAG 4888
Ncor1 GTGCCTGACCTGTTATCCTG 4889
Ncor1 GATTCCGCCACCGAGGAGAC 4890
Ncor1 GCCGTGGCTGTCCTGACTTG 4891
Ncor1 GGAACTCAGCGGAACGAATG 4892
Ncor1 GTCCAGTCATCACCATATTT 4893
Ncor1 GTAGGAGGGTCGCTGGGTTA 4894
Ncor1 GTTCCGCTGAGTTCCAAACC 4895
Ncor2 GAAGGAGAAGCCATGGAGGC 4896
Ncor2 GGCTTTGCCTTATAGAGACT 4897
Ncor2 GGAAGTTCATTTCAGCCTTT 4898
Ncor2 GGCAAGGTGTGCTGAGGTGG 4899
Ncor2 GTTAAAGATCTAAGGCAGAG 4900
Nccr2 GTAGGAGCCAGGGAGGACAA 4901
Nccr2 GCTGGGTAGCGGCACTACTC 4902
Ncor2 GAGCCCTCACATTGCCAGCC 4903
Ncor2 GAGTCATCCTCGCCATCCCA 4904
Ncor2 GTTCTAGCTTTAAGCCTGCC 4905
Neurod1 GCATAGTTCTTGGATACCTT 4906
Neurod1 GTTATCTCCGCTTGCCTGAC 4907
Neurod1 GTCGCCAGTTAGAGACTCCG 4908
Neurod1 GCGCATAAGAACAAGGCAGC 4909
Neurod1 GGTAGGAGCAGGTGACCGTT 4910
Neurod1 GGTCGGGCTACCTAACTCCA 4911
Neurod1 GTAACTGCAAGGCCCTTAGA 4912
Neurod1 GAACTATGCTGGGTAACAGT 4913
Neurod1 GTCAGAACCTTGCCTTCTAA 4914
Neurod1 GTGAAAGTATGTGTGTGTTG 4915
Neurod6 GTGCATCTGGGTACCAGGGA 4916
Neurod6 GATTAGAAGAGCCACTCTGG 4917
Neurod6 GTGTCTGTGTGTAAACCTGG 4918
Neurod6 GAGGGTTCATCCAGGATTCA 4919
Neurod6 GAGAGGGAAAGTTTCATATG 4920
Neurod6 GTAGAGCTAAAGTGAGTCTT 4921
Neurod6 GTTGTAACATGGGAGATCCA 4922
Neurod6 GTGTCACCGCTATGATTCTT 4923
Neurod6 GTGCTGCTGCCACATGTCAA 4924
Neurog1 GGCTGCTGGGAGTTGTGCAA 4925
Neurog1 GTGCACTACTGAATCCAAGA 4926
Neurog1 GTCAATCAGTAGCAGGCAAA 4927
Neurog1 GATTGGCCGGCGGTAATTAC 4928
Neurog1 GAATTGTCACAAGGTCAGAC 4929
Neurog1 GAGCAAGATTTCAGGAGAAG 4930
Neurog1 GCATAATTTATGCTCGCGGG 4931
Neurog1 GCTGTCACAGGGACAGAAAG 4932
Neurog1 GGCCCTGTATTTATTTCTTT 4933
Neurog1 GGCTGGCTGTCTATTAAGTC 4934
Neurog2 GAATAAAGGATGGGAACAGT 4935
Neurog2 GTTTCCTCTCAAGTCCAGCA 4936
Neurog2 GTATGACCTCTGCTCCGCTC 4937
Neurog2 GTCACGTACGTGTGCCAGAC 4938
Neurog2 GGACTTCAACACACGCCATC 4939
Neurog2 GATGAAAGGAGAGTCTTGGG 4940
Neurog2 GAGGGCTACGGAGCAGGATT 4941
Neurog2 GCCAAACAGACCCTTAGTGG 4942
Neurog2 GAAACGTGTCTATGACTGTT 4943
Neurog3 GGGAGGTGGTAGGATTGGGT 4944
Neurog3 GGATTCCGGACAAAGGGCAG 4945
Neurog3 GCCCATTAGTCTCACGGGAT 4946
Neurog3 GTGAAGCTGCTAGTCCTCTC 4947
Neurog3 GCATGGGAGGAAGCTATGGC 4948
Neurog3 GGGTAGACCTTCCTGTGAAC 4949
Neurog3 GAGGACAGAGTGACCAGAGA 4950
Neurog3 GCCCTTTAAGTCACTTTCCC 4951
Neurog3 GACAATGTCTTAAGGCTCAC 4952
Nf1 GTATCTTCCTATGTGGCTAA 4953
Nf1 GCCATGCATAGTGGTGTGAC 4954
Nf1 GGGAATTCTAGTCTCCAACT 4955
Nf1 GGCAATGACAGCCTACGCAC 4956
Nf1 GTCCTTCAAACTCTGGTTCT 4957
Nf1 GGCCCAGTGGTGATCCAAGT 4958
Nf1 GTCTCGGACTGTGATGGCTG 4959
Nf1 GATGGTGTGTGTGTGTGTGG 4960
Nf1 GAGCAAGAAGCCAGCAGTGA 4961
Nf1 GAAAGGATCCCACTTCCGGT 4962
Nfat5 GTGTCCTCCTAAGTACACCA 4963
Nfat5 GTGTTATGGGCCAACGTGTT 4964
Nfat5 GGGAATGGAGTTCCACAGCT 4965
Nfat5 GTGAATGGTCGAATTTACTC 4965
Nfat5 GCTAATGTCAATGACAGTTT 4967
Nfat5 GTGTAATGCACACGCGTGCG 4968
Nfat5 GTATCAGAIGTTCAGATGAA 4969
Nfat5 GCTGATCCCGGGCTGGGAAA 4970
Nfat5 GAGCTGATTTGTAGCCAGGA 4971
Nfat5 GACCTGGATGTCAGCCAGGA 4972
Nfatc1 GGGACGAAACGGGAAGGAAA 4973
Nfatc1 GCCGCTTGTTTATGTAAACC 4974
Nfatc1 GGACCCAGTACAGGGCTGAC 4975
Nfatc1 GACTCCTGGGAAAGAGTTGA 4976
Nfatc1 GACCAGCCGGACGCATTGAG 4977
Nfatc1 GGCTAACTTGAGCATCACGT 4978
Nfatc1 GCTAGATGCTGCTGGAAGAG 4979
Nfatc1 GACGGAACGGATTGGAGGGT 4980
Nfatc1 GGCCGTGGGAAAGCACCTTG 4981
Nfatc1 GTCTTGAGACAGCCAGACCC 4982
Nfatc2 GTCTCTTTGGAGGGTGGCCC 4983
Nfatc2 GCTTCTGCTGGTTTCTCTCC 4984
Nfatc2 GTTTGCACGCAGCTCCTGCA 4985
Nfatc2 GAGATAAAGCCAGCTTTGAT 4986
Nfatc2 GCGTAAACACATGCGTTGCC 4987
Nfatc2 GTTTGTAGAAACCTATGCCT 4988
Nfatc2 GGTGATGACTCACTAGCCCT 4989
Nfatc2 GCACAGTAAGAGGAGATTGG 4990
Nfatc3 GAAGTTGGTATGGAGGGATG 4991
Nfatc3 GGAGCTCATGTCGAGGAAGT 4992
Nfatc3 GGTGAAAGGAGTATGCATGT 4993
Nfatc3 GTGAAAGGAGTATGCATGTT 4994
Nfatc3 GCTACAGGAGTAGTAGAAAC 4995
Nfatc3 GCGATAGGTCGGTGAGGAGG 4996
Nfatc3 GATGGTGAGCAAGAGCTTTA 4997
Nfatc3 GCTACTAAGTGAGCCTCAGG 4998
Nfatc3 GCATTCAGATCAGCAGGAAG 4999
Nfatc3 GGGAACCCACGTAGGCCAAT 5000
Nf8tt4 GGAGAACAGACCCGGAAACT 5001
Nfatc4 GGTCTTCCAGACGAGGGAAG 5002
Nfatc4 GGCAGGGAGGAGAAGCTTGG 5003
Nfatc4 GGCTCTGAGCTGCTCTGTAG 5004
Nfatc4 GACAGTGAGGTGCCCTTTCT 5005
Nfatc4 GTGGTGGCTAAGAACTGCAA 5006
Nfatc4 GGAGATTTGCCAGGTTTATT 5007
Nfatc4 GAAACACTGCCCAGGATCAA 5008
Nfatc4 GAACTGCAAAGGCTCCTTGG 5009
Nfatc4 GTAACCTGAGAAGAACCCAA 5010
Nfe2 GATCCTCAAGGAGTGTGTTG 5011
Nfe2 GGGAATATGGAGGCAGGATG 5012
Nfe2 GACAGAGCTCTGCCTTGGGA 5013
Nfe2 GACACTATGGGAACTTGCTA 5014
Nfe2 GGGCAATTTCCGCCAGAACT 5015
Nfe2 GAAGTGGGCTGTAATGCCTC 5016
Nfe2 GCAAATTGGACTCAGATACC 5017
Nfe2 GTCTATGCAATCCACTCAGG 5018
Nfe2 GGGATGGCTTTATAGCAAGA 5019
Nfe2 GTGTCTCCTAAAGACCGACA 5020
Nfe2l1 GTGGGTAACTGGCATATCTG 5021
Nfe2l1 GAATTGTTGGTCATTGTGAT 5022
Nfe2l1 GGGTGGTGCAGTGAGAGTCC 5023
Nfe2l1 GACTAGCCATCGTCTTCTTA 5024
Nfe2l1 GTAAACTCCCTTTAGCTCCT 5025
Nfe2l1 GGCAGCCTAGGTAACAAGTT 5026
Nfe2l1 GCTAGGTAACAGGCGGTGGG 5027
Nfe2l1 GACCCTCAAGGACGGAATCT 5028
Nfe2l1 GGGTACCGGTTTCCGTTGCC 5029
Nfe2l1 GCAGCTAGGTAACAGGCGGT 5030
Nfe2l2 GATGTTTGTATGCGACAGTG 5031
Nfe2l2 GGTTCTGCAGGTCCAAATCA 5032
Nfe2l2 GTGAGACATCTAAGGCAAGA 5033
Nfe2l2 GAGATTACTGTATGACCTTG 5034
Nfe2l2 GGCATTCCTTTCTTCACCTC 5035
Nfe2l2 GAGAGGAGGATCAACAGTGG 5036
Nfe2l2 GGCAGTTAAAGAAGTATGTT 5037
Nfe2l2 GCTCTCCTGCCGACAGAGGT 5038
Nfe2l2 GGAGCTGCCACTCCCTGATT 5039
Nfe2l2 GGGCACGTGGGAGAAGTGGA 5040
Nfe2l3 GTGGTCCAGGTCACTACCAC 5041
Nfe2l3 GGAAAGTTGGAGAAGTTGGG 5042
Nfe2l3 GGGTGGGAGTGGAGGAAAGT 5043
Nfe2l3 GTGCTGCAATGCTGGCAGCT 5044
Nfe2l3 GCTGCCAGCATTGCAGCACT 5045
Nfe2l3 GTCACTACCACAGGGCTGCC 5046
Nfe2l3 GCAATCTCCAACAGCACACG 5047
Nfe2l3 GGAGAAGTTGGGAGGAGACA 5048
Nfe2l3 GGACACACTCATATCTGTTC 5049
Nfia GATAGGAGAGAAAGCAGGAG 5050
Nfia GCAAAGGCTGTAGTTGGAAC 5051
Nfia GCCAACTGAACCAGAAAGCA 5052
Nfia GTAGTTATATAGGCTAGTGT 5053
Nfia GATGCCGTAGAAATGAATTC 5054
Nfia GTTCACAATCTTGAGGAGGG 5055
Nfia GAAACAACAGTGGTTTAGCT 5056
Nfia GGATTTACCCTTCCTAACAA 5057
Nfia GCATAGGACATTCGGGATCC 5058
Nfia GTTTGCTTAAGCACATCCTG 5059
Nfib GTTTGAGCATTTCCCTAATG 5060
Nfib GCTCCATGTCGCCCTAGCTT 5061
Nfib GAAATAACCTCTCCCTGGGC 5062
Nfib GAACTTGATTCCCGGGACCC 5063
Nfib GGGTGCCAGGATTTCGCTGG 5064
Nfib GTTAAAGCTGGTATTATCAG 5065
Nfib GAACGCGCGTTTGCAGGAGG 5066
Nfib GAAGCAATAACAGTGTGGTG 5067
Nfib GAGAAAGCAGAGGTCTCAGG 5068
Nfib GAGAGAGTGCCCGCGCGAAA 5069
Nfic GGGCGCGCATCCAATCTGAC 5070
Nfic GTGCTGTCCCTAATATAGGG 5071
Nfic GACTTGTGAGTGGACACTGG 5072
Nfic GTCACTCACAGGCATCTCCT 5073
Nfic GTTGGCTCGGTAGTGACACC 5074
Nfic GCTGCTGCAGGGACTCAGGT 5075
Nfic GAGCTATCCATTTGTAGAGG 5076
Nfic GGTGGTTTGGTCAGTATCCG 5077
Nfic GACATGGGATGTGAGGGCTG 5078
Nfic GTCACTAACCCAGCAGGGTT 5079
Nfil3 GAAATGGGAGACAGAGCATC 5080
Nfil3 GTGCGTCACTGTCAGGAATA 5081
Nfil3 GATCCCTAAGTAGGTAGAAT 5082
Nfil3 GAAATGTCCCGCTCCTCTCC 5083
Nfil3 GCGTCCGGTGTTACACCCTG 5084
Nfil3 GAACTTGCCTGACTCACCCA 5085
Nfil3 GAGGATAAATCTCCTTTCAC 5086
Nfil3 GGTGGCAAGGTCCTTGAGCT 5087
Nfil3 GTTTCCCGGAGAGLCACAGA 5088
Nfix GGGAGGAATAGAGCAAATGA 5089
Nfix GCCATTGAACAGAAAGGCCA 5090
Nfix GGGAAAGTCCACACAAGTTG 5091
Nfix GGCCATGTTTGCAATTGTTT 5092
Nfix GGCGCTGCCTTCCCGTATAT 5093
Nfix GTGCTGCCCGTTTAGGGTAT 5094
Nfix GCGTCCATGCTCATAAACCA 5095
Nfix GTCCCAAACCTCTGAGATGG 5096
Nfix GTAGGACATAGAGAACTGTT 5097
Nfix GAAGGCAGAGGGCCTTTAGG 5098
Nfkb1 GACTCTCTAATATACAGTGT 5099
Nfkb1 GAAATTGTAACCTACGGGCC 5100
Nfkb1 GATTTGTAGAAGTTTGAGTG 5101
Nfkb1 GCCATTACTGAGGCGTTGAA 5102
Nfkb1 GATCGCTCCATAGAGCGGAC 5103
Nfkb1 GTCTCACTACTGAGTTCAAG 5104
Nfkb1 GTTGATTACAGGGCTCTTTA 5105
Nfkb1 GTTCTAACCAATGATGCCTA 5106
Nfkb1 GAGGCTCTGGAGAACTCCCA 5107
Nfkb1 GTTTGGTTGTTCCATGGCAG 5108
Nfkb2 GTTTGCTCCAGGCTGCGGAG 5109
Nfkb2 GATGTTTATTCTGTAAGTGG 5110
Nfkb2 GAGGGACCTCCTAGCTGGGA 5111
Nfkb2 GAGGACTTTAGATGACAGGC 5112
Nfkb2 GGGACCTCCTAGCTGGGAAG 5113
Nfkb2 GCTGTGCACAGGCAAGCTAA 5114
Nfkb2 GCCTTTCAAGTCAAATAGTT 5115
Nfkb2 GTGCGCTGTGAGTGCGTGTG 5116
Nfkb2 GGACTTTAGATGACAGGCTG 5117
Nfkb2 GACAGGGTGGTGTGAAACTT 5118
Nfkbib GTTCTTTGGGTAGAAAGGAA 5119
Nfkbib GCCGGCGGCCATATTGATAA 5120
Nfkbib GTACAGGCCTGAGAGCACGA 5121
Nfkbib GGAGATGCAGTGAAGGTAGG 5122
Nfkbib GAGCTGTOACAGCCTGCTGT 5123
Nfkbib GGCGAGACTGGACTGAAGGA 5124
Nfkbib GCATTCAGTGGTTGTAGGCA 5125
Nfkbib GTCGTATAAGTGAAGTGATA 5126
Nfkbib GGAGTACCGGGCAAACTCTG 5127
Nfkbib GGGAGACAGGATCTACCTGA 5128
Nfya GCGGTGTCAAATCCAGGAAG 5129
Nfya GCGCCCGCTCTCGGTAGTAA 5130
Nfya GCCTGCGTGGTATATAATTC 5131
Nfya GTAGCTATTCTGAAGAGGGA 5132
Nfya GCCCTCCTCCAAGCAGGGAA 5133
Nfya GTTGCCCTCCTTAGGGTAGG 5134
Nfya GGGTCATCCTTCACCTGCAA 5135
Nfya GAGAAGCAGGGTTGAAGCAG 5136
Nfya GCTTAGAAATAGGTGGGCAG 5137
Nfya GAGGATTGTCGAATGGGTGC 5138
Nfyb GCTACCATTCTCCCTTGTGG 5139
Nfyb GGTACAGGGTGGAAGTCGGC 5140
Nfyb GAGGAGGGTGTCCTAGAATT 5141
Nfyb GCTGTGTGCCTGAGGTGGCT 5142
Nfyb GGAAGGCCTTAAATGCACAG 5143
Nfyb GGTAGTAAGCCAACTTGGTA 5144
Nfyb GTAAATCTGGCTAGTAAGAA 5145
Nfyb GGATGAGAACGCCGGCCTCT 5146
Nfyb GGTAAATCTGGCTAGTAAGA 5147
Nfyc GCTGCGCACTACGCGTTGCT 5148
Myc GGAAACAGTCATGCTGTTAC 5149
Nfyc GTCTACAGTAATATCAGCTA 5150
Nfyc GGGTTGTGCATTGAGGCAAC 5151
Nfyc GCAGGAAGGGCTATAGCCCA 5152
Nfyc GTAATACATGCCTCTAATCT 5153
Nfyc GATAGTCTGTGATGTAATCT 5154
Nfyc GGAGCAGGAGGTCTTTCCCA 5155
Nfyc GAAACATTCTAGGGTGCTGA 5156
Nfyc GTAATTTCACTGCTTCTGAT 5157
Nhlh1 GGTCCTAGTCCTCCTTATCC 5158
Nhlh1 GACCCAGGTCCCGCAGACTT 5159
Nhlh1 GCACAGTGAGCTACAGTATA 5160
Nhlh1 GCAAAGATGAGGAGAGAGGA 5161
Nhlh1 GAAGAACCTTGAGAGACCAC 5162
Nhlh1 GTTCATCCCAACTCCCTACA 5163
Nhlh1 GGAACGAAGGCCTGAGGAGG 5164
Nhlh1 GTGTTAAGGCGTCATCCAAA 5165
Nhlh1 GGGAAGACAGAGAGGTAGGG 5166
Nhlh2 GGAGGTGGAAGATCCAAGAA 5167
Nhlh2 GGAGTGACGATGTGGGAGAG 5168
Nhlh2 GTCCCTGGTCACCCTCGTGT 5169
Nhlh2 GAAGGCTGGGCATCTGTGAG 5170
Nhlh2 GAGAGGAAGGTTTCCCAGCC 5171
Nhlh2 GAAAGCACAGCTGCTAGGAT 5172
Nh1h2 GGAACAGGGAGACAGGAGGT 5173
Nhlh2 GTGTCACAGCAAGCTGATGA 5174
Nhlh2 GGGAGCCAGGAGTGACGATG 5175
Nhlh2 GGGATACCTGGGTTGAGCAG 5176
Nhlh2 GAGGATGCTCAAACCATGGC 5177
Nkx1-1 GCGGGATCAGTTGGCTGTGG 5178
Nkx1-1 GGCGGGAGTCAAAGCCAGTG 5179
Nkxl-1 GGGAGCAAAGACCAAGATGG 5180
Nkx1-1 GTCAAAGCCAGTGAGGATGG 5181
Nkx1-1 GCTCATGTCAGAATATTGAG 5182
Nkx1-1 GAAGTGGAAGGAGGAGCAGA 5183
Nkx1-1 GTCCCACCTGGGTCCTTCAG 5184
Nkx1-1 GAGCCCGGCTTTGGAGGATG 5185
Nkx1-1 GACCTGCCTGCTGATGGGTA 5186
Nkx1-1 GTGGACTGTGCTCTGGCTCC 5187
Nkx1-2 GTAAGAAGTAGGAAGAGGAG 5188
Nkx1-2 GATTTGCACGCATTGTCCCT 5189
Nkx1-2 GGATATGTGTGTGTGTGCGG 5190
Nkx1-2 GGCTGTCCTCCCTGGAGACT 5191
Nkx1-2 GGCAAGGCTTTAAAGTCGGC 5192
Nkx1-2 GCCCTAAGCCTTCAGCTCTC 5193
Nkx1-2 GGCATCACATCCCAAAGCAG 5194
Nkx1-2 GCAATCAGGTCTGGCCTCTG 5195
Nkx1-2 GGACAATCGCTTGAGAAGCC 5196
Nkx1-2 GTTCTGTGTGATCGTGGCTG 5197
Nkx2-1 GTGTGCATACACACTGTATG 5198
Nkx2-1 GGATTAGCTAGGTTAGTGCT 5199
Nkx2-1 GTGCATACACACTGTATGTG 5200
Nkx2-1 GTGTCTAGGAGGCACCTGCC 5201
Nkx2-1 GGTGGTCATAGGAACACCAA 5202
Nkx2-1 GACTCAGTTCCACTCTGCAA 5203
Nkx2-1 GTGTCAGTGACTTAAATAAT 5204
Nkx2-1 GCGTTGTGTCTCTGTAGCTA 5205
Nkx2-1 GGCAAGTGGTAGATCTGGTT 5206
Nkx2-1 GCAGGAGGCACCAGCCATGA 5207
Nkx2-2 GTTGGGAGGGTAGAGGGCCT 5208
Nkx2-2 GGCCCAAGCAGCTGTGAGCT 5209
Nkx2-2 GGTTGAATGCCATGACAACT 5210
Nkx2-2 GTTCTGCTTCGCCTGGACTA 5211
Nkx2-2 GGTTTCCTTAATATTGTGGA 5212
Nkx2-2 GTCACAAGGCTCTAGAAACC 5213
Nkx2-2 GGTGAAGACCCAGAAATCCA 5214
Nkx2-2 GGGCGGTCTAGAGAAGGGAG 5215
Nkx2-2 GCTCTAGCAGTGGCAGGGTT 5216
Nkx2-2 GAGCACTGCTTGGTTGGACC 5217
Nkx2-3 GGTTCACCCACCCAGGGTTC 5218
Nkx2-3 GGAGAGGAGTGTTGTATCTG 5219
Nkx2-3 GAGCCGAATTGCCTCTTCTA 5220
Nkx2-3 GTTTCAGAAAGTTGAGGCCT 5221
Nkx2-3 GTCTGAIGGAGACCACCTTC 5222
Nkx2-3 GGGTGGGTGGAAGTCTCCAG 5223
Nkx2-3 GCCTGGCCTGAGTCAGTATT 5224
Nkx2-3 GCTGTCTGCTCCCTACCTGC 5225
Nkx2-3 GGGTACCCAACAAGGATCCC 5226
Nkx2-3 GGAAAGAAAGAACTGCGGGT 5227
Nkx2-4 GTGAGCTTGATAATAGACTC 5228
Nkx2-4 GTATGGTGCTCCTACTCTCA 5229
Nkx2-4 GGACTTGGGACACTTGAGCT 5230
Nkx2-4 GTGAGGAGAGGAAATGGGAA 5231
Nkx2-4 GTGTTGTGAGGAGAGGAAAT 5232
Nkx2-4 GCGAAGGATGGAGCTAGAAA 5233
Nkx2-4 GAGTCCAGGTTAAACTTTGG 5234
Nkx2-4 GCAAAGAATCTGCCTGTTGT 5235
Nkx2-5 GTTATGCTGAGTCTAAACGC 5236
Nkx2-5 GGGAGTCCTGTTAAGTGAAT 5237
Nkx2-5 GTTGTGCCTTTCAGAGCACA 5238
Nkx2-5 GGGTTCTGAGCTGAATGGAA 5239
Nkx2-5 GATCGGGCTAGAAAGGGTCT 5240
Nkx2-5 GCTGAGTCTAAACGCAGGGT 5241
Nkx2-5 GCGGCTGATTGCAGGAAAGG 5242
Nkx2-5 GATTGAAGATTGGTTTGTGT 5243
Nkx2-5 GTTGAAAGGGACAGAGACAA 5244
Nkx2-5 GATAGTCTCCCACTCCTGCA 5245
Nkx2-6 GGGTTTGGAGGGCTAGTTAG 5246
Nkx2-6 GAAGGCTCAGGGTTAGCACG 5247
Nkx2-6 GCTTAAGAGCAAAGACCTGG 5248
Nkx2-6 GAAAGCAGGGAGCCAGCCAG 5249
Nkx2-6 GGGTTAGCACGTGGTTTCTG 5250
NkX2-6 GTGCTATCTAGACCTGGGAG 5251
Nkx2-6 GAGATCAGTGGACCACTTGA 5252
Nkx2-6 GTACAACAGAGAGCTCCCGA 5253
Nkx2-6 GTGGTTTCTCTGGGAACCAA 5254
Nkx2-6 GGGAGAGTGCTGTTCAAACT 5255
Nkx2-9 GGGCTCAGTTGGGAGGACCA 5256
Nkx2-9 GCAATGTACAAGTCTTCCTT 5257
Nkx2-9 GGACCCTGAGTCTGGGACTC 5258
Nkx2-9 GTCTTGGGAGAAAGCAGGAG 5259
Nkx2-9 GTTTGAGCAGGGAAATGACC 5260
Nkx2-9 GGCACCGACTTGGGAGATGA 5261
Nkx2-9 GCTGAATTCGAACCTGACAA 5262
Nkx2-9 GAGCCAGAGGAAGACTAGAA 5263
Nkx2-9 GCACCGACTTGGGAGATGAA 5264
Nkx2-9 GCCAGAGGCAGAGGATGCAC 5265
Nkx3-1 GGGAAACCAGGAAAGGTTAA 5266
Nkx3-1 GGCATAGCCACTGCACCACT 5267
Nkx3-1 GGAATCAGAACTGAGCAGGC 5268
Nkx3-1 GGTTAAGGGCTCATCAGGGA 5269
Nkx3-1 GCAGTTACTCACTGTTTGGA 5270
Nkx3-1 GGGCTCCAGGTGACCCTCAA 5271
Nkx3-1 GTTGTCTAGATGTGTCCAGC 5272
Nkx3-1 GGTACAGTGCTATTTCAGTT 5273
Nkx3-2 GCTGGAGAAGGAACAGATTG 5274
Nkx3-2 GATGTTAATTTCAGAAGCTG 5275
Nkx3-2 GCGAGGAATTGGAAAGCATT 5276
Nkx3-2 GTGAGGAATGACACTCTGAT 5277
Nkx3-2 GTGAGCTCTGGACATGCTGA 5278
Nkx3-2 GTGTGATGGCCTGTGGACAT 5279
Nkx3-2 GGACCCTGCAGCATCTTCAT 5280
Nkx3-2 GCGAGGCGGACGACTTTGAC 5281
Nkx3-2 GGAATGACACTCTGATGGGA 5282
Nkx3-2 GTGGATTGGCTGGTTCCAAC 5283
Nkx6-1 GCCTGCCAGTCTCTAGGCTC 5284
Nkx6-1 GTGATAATGATCTAGGGAGT 5285
Nkx6-1 GGTTTGAAAGCAGCAAACCC 5286
Nkx6-1 GAGCTAATGGAGCAGGCAGG 5287
Nkx6-1 GCCTCTAGCCAGGTGCTGTC 5288
Nkx6-1 GTGTCACTGACTGCCCTTTC 5289
Nkx6-1 GGGTTTAGGTAGCAGAGGGC 5290
Nkx6-1 GGTCCAGACACCGTTGGAGG 5291
Nkx6-1 GATCATTATCACTTATGAGG 5292
Nkx6-1 GGGCAGTTGATACACCAGTG 5293
Nkx6-2 GGATGAATGAAGCGGGAGTG 5294
Nkx6-2 GAGACAGGGTAGGTGTGCTC 5295
Nkx6-2 GCTTAGTTCAGGGAAGAGCC 5296
Nkx6-2 GTGGGCTGTTGTGAACTTGT 5297
Nkx6-2 GTGCCTAGTGGTCCTGTCCT 5298
Nkx6-2 GGCGAACTATGAGACAGGGT 5299
Nkx6-2 GTTCAGGGAAGAGCCTGGGA 5300
Nkx6-2 GGGCGAATGGAAATTTGTTA 5301
Nkx6-2 GCATCTCCGTAGGTGGGCTG 5302
Nkx6-2 GGTCCTGGCGATTTAAGCAG 5303
Nkx6-3 GAGCAATCACTATTCTCTGG 5304
Nkx6-3 GAACAGAGCTACACAGAAAG 5305
Nkx6-3 GGCATTCCAcTGAAGAATGG 5306
Nkx6-3 GAGACCTAAGCAGGGCAGTC 5307
Nkx6-3 GATGAGCCAAGAAGAAGCGA 5308
Nkx6-3 GTCCACCAATGCCCAGATCC 5309
Nkx6-3 GGCTCATCTTTGGGAGTTCG 5310
Nkx6-3 GCGTCACATTCATTCCGACA 5311
Nkx6-3 GTAGGGACTGGAGGCTCCTG 5312
Nobox GAGAGACTTCTGACAGGAGT 5313
Nobox GGGTCAGCACTTCTAAGAAG 5314
Nobox GACTTCCAATAAGCTGCTGT 5315
Nobox GTTTAGTCTCCTCCAGGCCT 5316
Nobox GCTTCCCAAGGAAGGCCTTG 5317
Nobox GCCTGCTTGATGGAAAGGTA 5318
Nobox GGAGCAGAACAGCAATGGAA 5319
Nobox GCTCATATTCAAGGGTCAAG 5320
Nobox GCATGGTGCTCTTGCTGGTG 5321
Nov GCTGGAGAGTCAAGTCAAGC 5322
Nov GGTTGGAACTGTGAGGGCGG 5323
Nov GGAGCCATATGAGCTGGGCA 5324
Nov GGAGGCGTCCATCAGGTTAG 5325
Nov GCTGATTCTTGACCCTCTCC 5326
Nov GCAAAGTTTAGGCAGAGGTA 5327
Nov GTGCCATCTTGGAGTATTAG 5328
Nov GACTAAGCTTTGCCTAAAGG 5329
Nov GGGAAGAAAGGTGTAATTTA 5330
Npas1 GCTGGCAGAGCTTCCTGATG 5331
Npas1 GAGGCATAGAGACAAGACCT 5332
Npas1 GTGAGGATGCICCTACACTC 5333
Npas1 GGCATCCTGGAATTCTCACT 5334
Npas1 GTTACAGAACCTTCCAACAT 5335
Npas1 GCGATCGTGGTGGGACTCCA 5336
Npas1 GTGTGCTCACACGCATTCCA 5337
Npas1 GGGAACTATCCAGCAGGCAG 5338
Npas1 GTGACAGTTAAAGCTGCGCA 5339
Npas2 GTGTTTCTTCTCACCCAGGA 5340
Npas2 GAGGTGAGTCCTGCGCACTC 5341
Npas2 GATCTCTGGACGCCAGTAGA 5342
Npas2 GGCAGGGTTTGTAGGATGCT 5343
Npas2 GTCCTGGCTCATGGTGTTCT 5344
Npas2 GGCTAGACCAGCCGGAAGAG 5345
Npas2 GGTGCAGGTCCAGTTTGCAC 5346
Npas2 GATAGGTAGCCAGGAGCCAA 5347
Npas2 GTCAGAAACAAGCCTGAGGA 5348
Npas2 GTGAGAGTGAACGCTGTTCG 5349
Npas3 GAGCATTTCTACCTGGGTTA 5350
Npas3 ACAGTCACAGGGAGACTGGG 5351
Npas3 GAAAGATTGCATGGCACTAC 5352
Npas3 GGAGAACTTGATAGTTATCT 5353
Npas3 GTGAGCGGAAGAGTTGGTCT 5354
Npas3 GGGTTTCACTGAGCTAGGGT 5355
Npas3 GCCTGCACAGAGCAAAGGGC 5356
Npas3 GACGCCTGCCTTTCATTAGG 5357
Npas3 GTTTCCAGAATCATCAGGGT 5358
Npas3 GAAATAACCACCATCCGGGC 5359
Nr0b1 GATGCTGGATCGAGGAGCTG 5360
Nr0b1 GTTACTATCCTATGATGGTT 5361
Nr0b1 GAGGTCAGAGTCTAAGTTAA 5362
Nr0b1 GACCTTAAGGTGCAGGACTT 5363
Nr0b1 GGGACACTATCAGGAATAAA 5364
Nr0b1 GAACACTGAGCCAATGGGTA 5365
Nr0b1 GGTGACGAAGGCCAGCAATT 5366
Nr0b1 GGAACACTGAGCCAATGGGT 5367
Nr0b1 GTGGAGTGAAGAAGGAAAGG 5368
Nr0b1 GGATGCTGGATCGAGGAGCT 5369
Nr0b2 GAGACAAATGTCCAGGACAG 5370
Nr0b2 GAGAGAACAAACAGAGCTCA 5371
Nr0b2 GCGATAAGCCACTTCCAGGC 5372
Nr0b2 GTTCTACCCATACTGTAGGT 5373
Nr0b2 GAACCCTGGTCTTATGTGCA 5374
Nr0b2 GTCTCTAGIGGTCAGAGGTA 5375
Nr0b2 GGTTCTACCCATACTGTAGG 5376
Nr0b2 GTGACTGCTCCTTTCCATCA 5377
Nr0b2 GTATGGCCCACCTACAGTAT 5378
Nr0b2 GAGACTGTAAGGTCTTCCTG 5379
Nr1d1 GGGTAGGACTGGCATAGCAC 5380
Nr1d1 GCAAGGGCATGTGAATTCCT 5381
Nr1d1 GGGAGGAGCTAAGACAAACA 5382
Nr1d1 GAGGTAGTACTGGGACTAGG 5383
Nr1d1 GTGAGAAACACAGAGGCCTG 5384
Nr1d1 GTTGGCTGGGAGGAGGGAGA 5385
Nr1d1 GCTCCTCCCAGTTCCTCCCA 5386
Nr1d1 GATCTCAACGTGCCGGCTGC 5367
Nr1d1 GCTGGGAGGAAGGGAAGGAG 5388
Nr1d1 GGCAAGGGCATGTGAATTCC 5389
Nr1d2 GCTCAGAGTCCTGGAAAGCT 5390
Nr1d2 GCAGTAACCATGTGGGACCA 5391
Nr1d2 GGAGCCTGTATAGAGGAAGT 5392
Nr1d2 GAGGTCAGGACCGCTCGTTG 5393
Nr1d2 GGGATAAGCGGCTGCGAGAC 5394
Nr1d2 GGTCCACGGATTGGAAGAAG 5395
Nr1d2 GCGAGACAGGCTGGGAAGGA 5396
Nr1d2 GAGCAAACAACCTCTAGCAG 5397
Nr1d2 GTCACCTCCATGGTCCCACA 5398
Nr1h2 GATTCCCAACTGTCCATAAG 5399
Nr1h2 GCTGCGAGGAAAGTGAGGGA 5400
Nr1h2 GACAGACTTCCGGTCTGCCA 5401
Nr1h2 GGAGATGGCAAGATGGTTAC 5402
Nr1h2 GAGGTTATCTGAGGTTGGAC 5403
Nr1h2 GAGATGCTGGCCCTGGAAGC 5404
Nr1h2 GCGTCACTTCCGGAAGTAGG 5405
Nr1h2 GAGAGCTGCGAGGAAAGTGA 5406
Nr1h2 GGAAGTAACTTCAGAAGCCT 5407
Nr1h3 GAGGGAGCGCCAAGAGTAAA 5408
Nr1h3 GGGTGAAGACAGGCAGGTGC 5409
Nr1h3 GGGAAGGGTGAACATGGTTG 5410
Nr1h3 GAGCCTGTGAGCAGGAAACT 5411
Nr1h3 GACTGGGAACACGTGCAAGA 5412
Nr1h3 GAGGCTGCTGGGATTAGGGT 5413
Nr1h3 GAAGAGATTAGGGAGTCAGG 5414
Nr1h3 GAGGCAGAAGCTGAAGATGG 5415
Nr1h3 GACAGTGCTGCCTCTTCTAC 5416
Nr1h3 GAGGTGTCTTTGGGAGGAGG 5417
Nr1h4 GGAGGAGAAAGAAATGTATT 5418
Nr1h4 GGGATTCTCCAAACTGCTTC 5419
Nr1h4 GATACATGTAGAGGAGCTGA 5420
Nr1h4 GGATGTCAGCAAATTATGGC 5421
Nr1h4 GGAATAATTCCAACCATCAC 5422
Nr1h4 GGATCTTTACCTTTGTAACT 5423
Nr1h4 GCTGGAGGTTAAATGCCACA 5424
Nr1h4 GATCAAGGTGTTTACAAAGG 5425
Nr1h4 GTTCTTAGGAGATTAGAGGG 5426
Nr1h5 GTCCCAGCACCTATGTTAAT 5427
Nr1h5 GATCATTGCTGGCAAGGCAA 5428
Nr1h5 GAACTCCTCACTTACCCTTA 5429
Nr1h5 GATCTTGCTGCTGCGTGTCT 5430
Nr1h5 GAGAGCTGTACAGAAAGAAC 5431
Nr1h5 GAGCTGTACAGAAAGAACAG 5432
Nr1h5 GCACTGCTCTGCAGAGTGTC 5433
Nr1h5 GATGAAATCAAACGGACAGG 5434
Nr1h5 GTCACATCTTCTCTGACGGA 5435
Nr1i2 GGAGGAAATAGCTTCGAGAC 5436
Nr1i2 GAAGAGCATTTCTCTCCTTT 5437
Nr1i2 GAGTCACTCCCACGCATGGC 5438
Nr1i2 GGACAAGACGGGCTCCATTG 5439
Nr1i2 GGGACACTTATTTCCACGAG 5440
Nr1i2 GAGTGACTCAGGTCCTCTCT 5441
Nr1i2 GCCAGAGAACCAGAGAGAAT 5442
Nr1i2 GGGAAATTGAACAAACCAGA 5443
Nr1i2 GCTAGCTCGGGTGCTGGACT 5444
Nr1i2 GGAGTGACTCAGGTCCTCTC 5445
Nr1i3 GTGTTGGTTGGTGGCAGATG 5446
Nr1i3 GTGCCTGCTGAGGTCAGAAG 5447
Nr1i3 GTAGCATTGGGCAAGCTATG 5448
Nr1i3 GTATCAGGGTTGGAGCCTGG 5449
Nr1i3 GACCTCAGCAGGCACAAATA 5450
Nr1i3 GCATGGATCCTGAATAAGCC 5451
Nr1i3 GGATCCCACTTTCTTACGTG 5452
Nr1i3 GCCACCAACCAACACTTCTC 5453
Nr1i3 GGGATCCCACTTTCTTACGT 5454
Nr2c1 GCAGGAACTGTTAACTATCT 5455
Nr2c1 GGAGTCTGTGTAGGATAACA 5456
Nr2r1 GACACCAGAGTTGCAGGTAT 5457
Nr2c1 GTACCCTTCTCCCTCGAATC 5458
Nr2c1 GCCAGTGAGGTTCATCTAAA 5459
Nr2c1 GCAGAATCCTGAGCCGGAGG 5460<
